CRYSTALLINE SULFATE SALT FORMS OF AN 8-AZABICYCLO[3.2.1]OCTANE COMPOUND

FORMES DE SEL DE SULFATE CRISTALLIN D'UN COMPOSE 8-AZABICYCLO[3.2.1]OCTANE

English Abstract

The invention provides a crystalline sulfate salt of 3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide or a solvate thereof. The invention also provides pharmaceutical compositions comprising such crystalline salt forms, methods of using such crystalline salt forms to treat diseases associated with mu opioid receptor activity, and processes useful for preparing such crystalline salt forms.

French Abstract

L'invention concerne un sel de sulfate cristallin de 3-endo-(8-{2-[cyclohexylméthyl-((S)-2,3-dihydroxy-propionyl)amino]éthyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide ou un solvate de celui-ci. L'invention fournit également des compositions pharmaceutiques comprenant de telles formes de sel cristallin, des procédés d'utilisation de telles formes de sel cristallin pour traiter des maladies associées à l'activité de récepteur opioïde mu et des procédés utiles pour préparer de telles formes de sel cristallin.

Claims

WHAT IS CLAIMED IS:

1. A crystalline salt form which is the sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-
((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-
yl)benzamidewherein the crystalline salt form is characterized by an x-ray
powder
diffraction pattern having two or more diffraction peaks at 20 values selected
from
6.58~0.20, 7.52~0.20, 9.35~0.20, 14.69~0.20, 16.01~0.20, 17.45~0.20,
17.99~0.20,
18.62~0.20, 19.76~0.20, 21.11~0.20, 22.07~0.20, 23.18~0.20, 23.74~0.20,
24.56~0.20, 25.63~0.20, 26.45~0.20, 27.86~0.20, 28.31~0.20, 29.54~0.20,
30.59~0.20, 31.58~0.20, 33.89~0.20, and 36.02~0.20.
2. The crystalline salt form of Claim 1 wherein the x-ray powder
diffraction pattern
comprises two or more diffraction peaks at 2.THETA. values selected from
14.69~0.20,
16.01~0.20, 21.11~0.20, 22.07~0.20, and 23.18~0.20.
3. The crystalline salt form of Claim 1, wherein the crystalline salt form
is characterized
by an x-ray powder diffraction pattern in which the peak positions are as
shown in
Figure 1.
4. The crystalline salt form of Claim 1, wherein the crystalline salt form
is characterized
by a differential scanning calorimetry trace recorded at a heating rate of 10
°C per
minute which shows a maximum in endothermic heat flow at a temperature between

about 190 °C and about 205 °C.
5. The crystalline salt form of Claim 1, wherein the crystalline salt form
is characterized
by a differential scanning calorimetry trace as shown in Figure 2.
6. A crystalline salt form which is the hydrate of a sulfate salt of 3-endo-
(8-{2-
[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-
bicyclo[3.2.1]oct-3-yl)benzamide wherein the crystalline salt form is
characterized by
an x-ray powder diffraction pattern having two or more diffraction peaks at
2.THETA. values
selected from 9.41~0.20, 9.98~0.20, 15.17~0.20, 16.70~0.20, 18.59~0.20,
19.46~0.20, 19.91~0.20, 20.63~0.20, 21.35~0.20, 21.89~0.20, 23.00~0.20,
24.20~0.20, 25.40~0.20, 26.03~0.20, 27.44~0.20, 28.46~0.20, 29.45~0.20,
31.22~0.20, 31.82~0.20, 33.17~0.20, 33.56~0.20, and 36.89~0.20.

43


7. The crystalline salt form of Claim 6 wherein the x-ray powder
diffraction pattern
comprises two or more diffraction peaks at 2.THETA. values selected from
16.70~0.20,
18.59~0.20, 19.46~0.20, 19.91~0.20, 23.00~0.20, and 24.20~0.20.
8. The crystalline salt form of Claim 6, wherein the crystalline salt form
is characterized
by an x-ray powder diffraction pattern in which the peak positions are as
shown in
Figure 4.
9. The crystalline salt form of Claim 6, wherein the crystalline salt form
is characterized
by a differential scanning calorimetry trace as shown in Figure 5.
10. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and
the crystalline salt form as defined in any one of Claims 1 to 9.
11. A process for preparing a crystalline sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-
((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-
yl)benzamide
as defined in Claim 1, the process comprising:
(a) contacting a protected precursor to 3-endo-(8-{2-[cyclohexylmethyl-((S)-
2,3-
dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide, in
which the hydroxy groups are protected, with sulfuric acid to form a first
reaction mixture;
(b) isolating an intermediate grade solid sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-
bicyclo[3.2.1]oct-3-yl)benzamide from the first reaction mixture;
(c) dispersing the intermediate grade solid sulfate salt in a diluent
comprising
methanol to form a second reaction mixture; and
(d) isolating the crystalline sulfate salt from the second reaction
mixture.
12. The process of Claim 11 wherein the protected precursor is (S)-2,2-
dimethyl-
[1,3]dioxolane-4-carboxylic acid {2-[3-(3-carbamoyl-phenyl)-8-
azabicyclo[3.2.1]oct-
8-yl]ethyl}cyclohexylmethyl-amide.
13. The process of Claim 12 wherein the diluent comprising methanol further
comprises
up to 25 % water.

44


14. The process of Claim 12 wherein the diluent comprising methanol
comprises
between about 5 % and about 15 % water.
15. A process for preparing a crystalline sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-
((S)-2,3 -dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-
yl)benzamide
as defined in Claim 1, the process comprising:
(a) contacting 3-endo-(8-{24cyclohexylmethyl-((S)-2,3-dihydroxy-
propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide with sulfuric
acid to form a reaction mixture; and
(b) isolating the crystalline sulfate salt from the reaction mixture.
16. A process for preparing a crystalline sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-
((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-
yl)benzamide
as defined in Claim 1, the process comprising:
(a) dispersing a crystalline hydrate of the sulfate salt of 3-endo-(8-{2-
[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-
bicyclo[3.2.1]oct-3-yl)benzamide in a diluent comprising methanol to form a
reaction mixture; and
(b) isolating the crystalline sulfate salt from the reaction mixture.
17. The process of Claim 16 wherein the diluent comprising methanol further
comprises
up to 25 % water.
18. A bisulfite adduct of N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid
benzyl ester.
19. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for manufacture
of a medicament for the treatment of a disease or medical condition in a
mammal
ameliorated by treatment with a mu opioid receptor antagonist.
20. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for manufacture
of a medicament for the treatment of opioid-induced bowel dysfunction or post-
operative ileus.



21. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for manufacture
of a medicament for enhancing motility of the gastrointestinal tract in a
mammal
22. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for manufacture
of a medicament for antagonizing a mu opioid receptor in a mammal.
23. A crystalline salt form as defined in any one of Claims 1 to 9 for use
in the treatment
of a disease or medical condition in a mammal ameliorated by treatment with a
mu
opioid receptor antagonist.
24. A crystalline salt form as defined in any one of Claims 1 to 9 for use
in the treatment
of opioid-induced bowel dysfunction or post-operative ileus.
25. A crystalline salt form as defined in any one of Claims 1 to 9 for use
in enhancing
motility of the gastrointestinal tract in a mammal.
26. A crystalline salt form as defined in any one of Claims 1 to 9 for use
in antagonizing a
mu opioid receptor in a mammal.
27. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for the treatment
of a disease or medical condition in a mammal ameliorated by treatment with a
mu
opioid receptor antagonist.
28. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for the treatment
of opioid-induced bowel dysfunction or post-operative ileus.
29. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for enhancing
motility of the gastrointestinal tract in a mammal
30. Use of a crystalline salt form as defined in any one of Claims 1 to 9
for antagonizing a
mu opioid receptor in a mammal.

46

Description

CA 02678073 2014-05-13
WO 2008/106159 PC
T/US2008/002583
CRYSTALLINE SULFATE SALT FORMS OF AN
8-AZABICYCLO[3.2.1]0CTANE COMPOUND
BACKGROUND OF THE INVENTION
Field of the Invention
The invention is directed to crystalline salt forms of an 8-
azabicyclo[3.2.1]octane
compound which are useful as mu opioid receptor antagonists. The invention is
also
directed to pharmaceutical compositions comprising such crystalline compounds,
methods
of using such compounds for treating or ameliorating medical conditions
mediated by mu
opioid receptor activity, and processes useful for preparing such compounds.
State of the Art
Commonly-assigned U.S. Provisional Application Nos. 60/777,962, filed on
March 1, 2006, and 60/841,028, filed on August 30, 2006, and U.S. Application
Serial
No. 11/711,961 disclose 8-azabicyclo[3.2.1]octane compounds that are mu opioid
receptor antagonists that are expected to be useful for treating or
ameliorating medical
conditions mediated by mu opioid receptor activity. In particular, the
compound 3-endo-
(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propionyl)amino]ethyl) -8-aza-
bicyclo[3.2.1]oct-3-yl)benzamide sulfate is specifically disclosed in these
applications as
demonstrating mu opioid receptor antagonist activity.
The chemical structure of 3-endo-(8- {2-[cyclohexylmethyl-((5)-2,3-dihydroxy-
propionyl)aminolethyl}-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide (hereinafter
compound 1)
is shown below:
1

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0
N
= NH,
H6. OH
1
To effectively use this compound as a therapeutic agent, it would be desirable
to
have a solid-state salt form that can be readily manufactured and that has
acceptable
chemical and physical stability. For example, it would be highly desirable to
have a salt
form that is thermally stable, for example at temperatures exceeding about 175
C or
about 180 C, and is not hygroscopic nor deliquescent, thereby facilitating
processing and
storage of the material. Crystalline solids are generally preferred over
amorphous forms,
for enhancing purity and stability of the manufactured product.
No crystalline salt forms of compound 1 have previously been reported.
Accordingly, a need exists for a stable, crystalline salt form of compound 1
that is neither
hygroscopic nor deliquescent, and exhibits favorable thermal stability.
SUMMARY OF THE INVENTION
The present invention provides a crystalline sulfate salt of 3-endo-(8- {2-
[cyclohexylmethy14(5)-2,3-dihydroxy-propionyDamino]ethyl} -8-aza-
bicyclo[3.2.1]oct-3-
yl)benzamide or a solvate thereof. In one aspect, the crystalline salt form of
the invention
is a crystalline sulfate salt of compound 1. In another aspect, the
crystalline salt form of
the invention is a crystalline hydrate of the sulfate salt of compound 1.
Surprisingly, the crystalline sulfate salt of the invention has been found to
exhibit
no significant thermal events below a melting temperature in the range of
about 190 to
about 205 C and to exhibit a weight change of less than about 0.3 % when
exposed to a
range of relative humidity between about 2 % and about 90 % at room
temperature.
Furthermore, neither the crystalline sulfate salt of the invention nor the
hydrate thereof is
deliquescent when exposed to up to about 90 % relative humidity at room
temperature.
Among other uses, the crystalline salt forms of the invention are expected to
be
useful for preparing pharmaceutical compositions for treating or ameliorating
medical
conditions mediated by mu opioid receptor activity. Accordingly, in another of
its
composition aspects, the invention provides a pharmaceutical composition
comprising a
pharmaceutically-acceptable carrier and a crystalline sulfate salt of 3-endo-
(8- {2-
2

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[cyclohexylmethy14(5)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-
bicyclo[3.2.1]oct-3-
y1)benzamide or a solvate thereof.
The invention also provides a method of treating or ameliorating a disease or
condition ameliorated by treatment with a mu opioid receptor antagonist, e.g.
a disorder of
reduced motility of the gastrointestinal tract, the method comprising
administering to the
mammal, a therapeutically effective amount of a crystalline sulfate salt of 3-
endo-(8- {2-
[cyclohexylmethy145)-2,3-dihydroxy-prop ionyDamino] ethyl) -8-aza-bicyclo [3
.2.1] o ct-3-
yl)benzamide or a solvate thereof.
The invention further provides a method of treating opioid-induced bowel
dysfunction or post-operative ileus, the method comprising administering to
the mammal,
a therapeutically effective amount of a crystalline sulfate salt of 3-endo-(8-
{2-
[cyclohexylmethyl4S)-2,3-dihydroxy-propionyDamino]ethyl)-8-aza-
bicyclo[3.2.1]oct-3-
yl)benzamide or a solvate thereof.
In another method aspect, the invention provides a process for preparing a
crystalline sulfate salt of the invention, the process comprising contacting 3-
endo-(8- {2-
[cyclohexylmethy149-2,3-dihydroxy-propionyDamino]ethyl)-8-aza-
bicyclo[3.2.1]oct-3-
yl)benzamide with sulfuric acid to form a reaction mixture, and isolating the
crystalline
sulfate salt from the reaction mixture.
The invention provides an additional process for preparing a crystalline
sulfate salt
of the invention, the process comprising dispersing a crystalline hydrate of
the sulfate salt
of 3-endo-(8- {2-[cyclohexylmethyl4S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-
aza-
bicyclo[3.2.1]oct-3-yl)benzamide in a diluent comprising methanol to form a
reaction
mixture, and isolating the crystalline sulfate salt from the reaction mixture
In yet another method aspect, the invention provides a process for preparing a
crystalline sulfate salt of compound 1, the process comprising: (a) contacting
a protected
precursor to compound 1 in which the hydroxy groups are protected, with
sulfuric acid to
form a first reaction mixture; (b) isolating an intermediate grade solid
sulfate salt of
compound 1 from the first reaction mixture; (c) dispersing the intermediate
grade solid
sulfate salt in a diluent comprising methanol to form a second reaction
mixture; and
(d) isolating the crystalline sulfate salt from the second reaction mixture.
In a related composition aspect, the invention provides the N-cyclohexylmethyl-

(2-oxoethyl)-carbamic acid benzyl ester bisulfite adduct, which is useful for
the
preparation of the above protected precursor to compound 1.
3

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The invention also provides a crystalline sulfate salt of the invention as
described
herein for use in therapy or as a medicament, as well as the use of a
crystalline sulfate salt
of the invention in the manufacture of a medicament, especially for the
manufacture of a
medicament for treating a disorder associated with mu opioid receptor activity
in a
mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
Various aspects of the present invention are illustrated by reference to the
accompanying drawings.
Figure 1 shows an x-ray powder diffraction (XRPD) pattern of a crystalline
sulfate
salt of 3-endo-(8- {24cyclohexylmethyl4S)-2,3-dihydroxy-propionypamino]ethyl) -
8-
aza-bicyclo[3.2.1]oct-3-yl)benzamide of the invention.
Figure 2 shows a differential scanning calorimetry (DSC) trace (right side
vertical
axis) and a thermal gravimetric analysis (TGA) trace (left side vertical axis)
for a
crystalline sulfate salt of 3-endo-(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-

propionyl)amino]ethyl)-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide of the
invention.
Figure 3 shows a dynamic moisture sorption (DMS) trace for a crystalline
sulfate
salt of 3-endo-(8- {24cyclohexylmethyl-((5)-2,3-dihydroxy-
propionypamino]ethyl} -8-
aza-bicyclo[3.2.1]oct-3-yObenzamide of the invention.
Figure 4 shows an x-ray powder diffraction (XRPD) pattern of a crystalline
hydrate of a sulfate salt of 3-endo-(8-12-[cyclohexylmethyl-((5)-2,3-dihydroxy-

propionyl)amino]ethyl)-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide of the
invention.
Figure 5 shows a differential scanning calorimetry (DSC) trace (right side
vertical
axis) and a thermal gravimetric analysis (TGA) trace (left side vertical axis)
for a
crystalline hydrate of a sulfate salt of 3-endo-(8- {24cyclohexylmethyl-((S)-
2,3-dihydroxy-
propionypamino]ethyl)-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide of the invention.
Figure 6 shows a dynamic moisture sorption (DMS) trace for a crystalline
hydrate
of a sulfate salt of 3 -endo-(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-
propionyl)amino]ethyll-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide of the
invention.
4

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DETAILED DESCRIPTION OF THE INVENTION
The invention provides a crystalline sulfate salt of 3-endo-(8- {2-
[cyclohexylmethyl-((S)-2,3-dihydroxy-prop ionyDamino] ethyl} -8-aza-bicyc lo
[3.2.1] o ct-3 -
yObenzamide or a solvate thereof.
Definitions
When describing the compounds, compositions and methods of the invention, the
following terms have the following meanings, unless otherwise indicated.
The term "therapeutically effective amount" means an amount sufficient to
effect
treatment when administered to a patient in need of treatment.
The term "treatment" as used herein means the treatment of a disease,
disorder, or
medical condition in a patient, such as a mammal (particularly a human) which
includes:
(a) preventing the disease, disorder, or medical condition from
occurring, i.e.,
prophylactic treatment of a patient;
(b) ameliorating the disease, disorder, or medical condition, i.e.,
eliminating or
causing regression of the disease, disorder, or medical condition in a
patient, including counteracting the effects of other therapeutic agents;
(c) suppressing the disease, disorder, or medical condition, i.e., slowing
or
arresting the development of the disease, disorder, or medical condition in
a patient; or
(d) alleviating the symptoms of the disease, disorder, or medical condition
in a
patient.
The term "solvate" means a complex or aggregate formed by one or more
molecules of a solute, i.e. a compound of the invention or a pharmaceutically-
acceptable
salt thereof, and one or more molecules of a solvent. Such solvates are
typically
crystalline solids having a substantially fixed molar ratio of solute and
solvent.
Representative solvents include by way of example, water, methanol, ethanol,
isopropanol, acetic acid, and the like. When the solvent is water, the solvate
formed is
specifically termed a hydrate.
The term "crystalline sulfate salt", or alternatively "crystalline sulfate
salt
(anhydrous form)" or "anhydrous sulfate salt", as used herein means a
crystalline solid
that does not include a substantially fixed molar fraction of solvent
molecules in the
5

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crystal lattice, i.e. one that is not a solvate or hydrate. Solvates, or
specifically hydrates,
of the invention are identified explicitly.
It must be noted that, as used in the specification and appended claims, the
singular forms "a", "an", "one", and "the" may include plural references,
unless the
content clearly dictates otherwise.
Active Agent
The active agent in the present salt forms, i.e. compound 1, is designated 3-
endo-
(8- {2-[cyclohexylmethyl-((5)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-
bicyclo[3.2.1]oct-3-yObenzamide. Alternatively, using the IUPAC conventions as
implemented in AutoNom software, (MDL Information Systems, GmbH, Frankfurt,
Germany), the compound is denoted 34(1R,3R,5S)-8-124cyclohexylmethyl-((5)-2,3-
dihydroxy-propionyl)amino]ethyl}-8-azabicyclo[3.2.1]oct-3-y1)benzamide. The
name
used herein therefore corresponds to the IUPAC notation with the endo
orientation of the
substituted phenyl group with respect to the 8-azabicyclo[3.2.1]octane group
indicated
explicitly. In yet other common nomenclatures, the "((5)-2,3-dihydroxy-
propionyl)amino" portion of the molecule is variously designated as ((S)-2,3-
dihydroxy-1-
oxopropyl)amino or ((5) -2,3-dihydroxypropanamido)
Salt Forms of the Invention
In one aspect, the invention provides crystalline 3-endo-(842-
[cyclohexylmethyl-
((S)-2,3-dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.1]oct-3-
y1)benzamide
sulfate.
In one aspect, the crystalline sulfate salt of the present invention is
characterized
by an x-ray powder diffraction (XRPD) pattern having two or more diffraction
peaks,
including three or more and four or more diffraction peaks, at 20 values
selected from
6.580.20, 7.520.20, 9.350.20, 14.690.20, 16.010.20, 17.450.20, 17.990.20,
18.620.20, 19.760.20, 21.110.20, 22.070.20, 23.180.20, 23.740.20, 24.560.20,
25.630.20, 26.450.20, 27.860.20, 28.310.20, 29.540.20, 30.590.20, 31.580.20,
33.890.20, and 36.02 0.20. In particular, in this aspect, the crystalline form
is
characterized by an x-ray powder diffraction pattern having two or more
diffraction peaks,
including three or more and four or more diffraction peaks, at 20 values
selected from
14.690.20, 16.010.20, 21.110.20, 22.070.20, and 23.180.20.
6

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As is well known in the field of powder x-ray diffraction, peak positions of
XRPD
spectra are relatively less sensitive to experimental details, such as details
of sample
preparation and instrument geometry, than are the relative peak heights. Thus,
in one
aspect, a crystalline sulfate salt of compound 1 is characterized by an x-ray
powder
diffraction pattern in which the peak positions are substantially in
accordance with those
shown in Figure 1.
The crystalline sulfate structure has been further characterized by single
crystal
x-ray diffraction analysis, providing the following lattice parameters: unit
cell is
orthorhombic with dimensions a = 6.8239 A, b = 16.2275 A, c = 24.2021 A,
a =13 = y = 900; cell volume (V) of 2680.0 A3; calculated density is 1.38
g/cm3; space
group is P212121(#19). The resulting molecular structure confirms the chemical

composition is that of the sulfate salt of compound 1 in a 1:1 molar ratio of
sulfate
counterion to compound 1 and that the asymmetric unit cell does not contain
water or
other solvent molecules. X-ray powder diffraction peaks predicted from the
derived
atomic positions are in excellent agreement with the observed XRPD pattern.
In another aspect, the crystalline sulfate salt of the present invention is
characterized by its behavior when exposed to high temperature. As
demonstrated in
Figure 2, the differential scanning calorimetry (DSC) trace of a highly
crystalline sample
exhibits a peak in endothermic heat flow, identified as a melt transition, in
the range of
about 190 C to about 205 C. The thermal gravimetric analysis (TGA) trace
shows no
significant weight loss at temperatures below the melting point. Thermal
decomposition
occurs approximately upon melting.
In yet another aspect a crystalline sulfate salt is characterized by its
infrared
absorption spectrum which shows significant absorption bands at about 430,
590, 639,
705, 867, 1036, 1053, 1105, 1171, 1231, 1277, 1375, 1391, 1452, 1476, 1553,
1596,
1639, 1664, 2852, 2907, 2928, 2967, 3168, and 3357 cm-1

.
A crystalline sulfate salt of compound 1 has been demonstrated to have a
reversible sorption/desorption profile with an exceptionally low level of
hygroscopicity
(i.e., less than about 0.3 % weight gain in the humidity range of 2 % relative
humidity to
90 % relative humidity at room temperature) as shown in Figure 3.
Additionally, the crystalline sulfate salt of compound 1 has been found to be
stable
upon exposure to elevated temperature and humidity. After storage for 4 weeks
at 40 C
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and 75 % relative humidity, analysis by HPLC showed no chemical degradation
and there
were no detectable changes in the DSC, TGA, or XRPD results.
In another aspect, the invention provides a crystalline hydrate of a sulfate
salt of
compound 1.
In one aspect, a crystalline hydrate of a sulfate salt of the present
invention is
characterized by an x-ray powder diffraction (XRPD) pattern having two or more

diffraction peaks, including three or more and four or more diffraction peaks,
at 20 values
selected from 9.410.20, 9.980.20, 15.170.20, 16.700.20, 18.590.20, 19.460.20,
19.910.20, 20.630.20, 21.350.20, 21.890.20, 23.000.20, 24.200.20, 25.400.20,
26.030.20, 27.440.20, 28.460.20, 29.450.20, 31.220.20, 31.820.20, 33.170.20,
33.560.20 and 36.89 0.20. In particular, in this aspect, the crystalline form
is
characterized by a x-ray powder diffraction pattern having two or more
diffraction peaks,
including three or more and four or more diffraction peaks, at 20 values
selected from
16.700.20, 18.590.20, 19.460.20, 19.910.20, 23.000.20, and 24.200.20.
In another aspect, a crystalline hydrate of a sulfate salt of compound 1 is
characterized by an x-ray powder diffraction pattern in which the peak
positions are
substantially in accordance with those shown in Figure 4.
The crystalline hydrate of a sulfate salt of the present invention is also
characterized by its differential scanning calorimetry (DSC) trace which
exhibits two
endothermic events: a first peak in endothermic heat flow in the range of
about 125 to
about 133 C and a second peak in the range of about 178 to about 183 C when
analyzed
at a heating rate of 10 C per minute as illustrated in Figure 5. The thermal
gravimetric
analysis (TGA) trace shows a first thermal event between about 60 and about
140 C and
a second thermal event between about 140 and about 190 C. Analysis by TGA
coupled
with IR of the material vaporized in the first thermal event is consistent
with a hydrate
composition having about one mole of water per mole of compound 1 sulfate.
Surprisingly, a crystalline hydrate of a sulfate salt of compound 1 has
demonstrated low hygroscopicity. As illustrated in Figure 6 the crystalline
hydrate
exhibits a reversible sorption/desorption profile at room temperature over the
entire range
of about 2 % to about 90 % relative humidity with less than about 0.3 % weight
gain over
the entire range in relative humidity.
These properties of the salt forms of this invention are further illustrated
in the
Examples below.
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Synthetic Procedures
The active agent, 3-endo-(8-{2-{cyclohexylmethyl-((S)-2,3-dihydroxy-
propionyl)amino]ethyll-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide, can be prepared
from
readily available starting materials using the procedures described in the
Examples below,
or using the procedures described in the commonly-assigned U.S. applications
listed in
the Background section of this application.
In one method of preparation, a crystalline sulfate salt of the invention is
prepared
by contacting 3-endo-(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-
propionyl)amino]ethy1}-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide with about 0.5
to about
1.5 molar equivalents, including about 1 molar equivalent, of sulfuric acid.
Generally,
this reaction is conducted in an inert diluent at a temperature ranging from
about 0 C to
about 65 C, including about 60 to about 65 C. Suitable inert diluents
include, for
example, methanol, toluene, dichloromethane, and combinations such as toluene
and
acetonitrile, dichloromethane and acetonitrile, and toluene, acetonitrile, and
water, in
addition to a methanol and water combination comprising about 10 % water.
Using these
diluents, a reaction mixture with a concentration of between about 5 and about
400 mg/mL, including between about 50 and about 100 mg/mL, is prepared and
held for
between about 2 and about 24 hours, with optional agitation. The mixture may
be cooled
to between about 5 and about 20 C during the holding period.
Upon completion of the reaction, a crystalline salt of the invention is
isolated from
the reaction mixture by any conventional means, such as filtration,
concentration,
centrifugation, and the like.
Alternatively, the crystalline sulfate of the invention is prepared by
recrystallization of the hydrate form. The crystalline hydrate is dispersed in
an inert
diluent as described above at a concentration of between about 5 and about 400
mg/ml.
Methanol, or a methanol:water combination, in a ratio of from about 3:1 to
about 9:1, are
particularly useful diluents for this reaction. The reaction mixture is
maintained at
temperatures in the range of about 0 to about 65 C, typically with agitation,
for between
about 1 and about 24 hours, including between about 1 and about 6 hours.
Typically the
reaction mixture is cooled from about 65 C to between about 5 and about 20 C
during
the holding period. To improve the yield, the volume of the solution may be
reduced by
about 50 % before holding the reaction mixture for a time period of between
about 1 and
9

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about 24 hours, including between about 1 and about 6 hours, at a temperature
of between
about 5 and about 20 C. The resulting crystals are recovered conventionally.
Both the crystalline sulfate salt and the crystalline hydrate of the sulfate
salt of
compound 1 are advantageously prepared from the protected precursor of
compound 1.
As described in the examples below, to prepare the active agent, the protected
aldehyde 2,
N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester, regenerated from
its
bisulfite adduct 3, is coupled with 3-endo-(8-azabicyclo[3.2.1]oct-3-y1)-
benzamide
hydrochloride 4 to provide protected intermediate 5, which is deprotected to
provide 3-
endo- {842-(cyclohexylmethylamino)ethyl]-8-aza-bicyclo[3.2.1]oct-3-
y1}benzamide 6.
NH,
N NCI HN
OH
74,0 0 0
ONa 4
3 2
0
N
NH2 H2
*
5 6
Reaction of intermediate 6 with lithium (48)-2,2-dimethy1-1,3-dioxolane-4-
carboxylate 7
provides the protected intermediate (5)-2,2-dimethy141,3]dioxolane-4-
carboxylic acid {2-
[3-(3-carbamoyl-pheny1)-8-azabicyclo[3.2.1]oct-8-yl]ethyl}cyclohexylmethyl-
amide 8
0
Li0 0 NH2
0
N NH2
7 /
N
=laõN
0
6-7c
6 8
Protected intermediate 8 is contacted with between about 0.8 and about 1.3
equivalents, typically about 1 to about 1.2 equivalents, of aqueous sulfuric
acid in an inert
diluent, such as ethyl acetate or isopropyl acetate, at a temperature of
between about 20
and about 30 C. A second diluent, which is miscible with the reaction
mixture, and in

CA 02678073 2009-08-13
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which the product is less soluble, is typically included in the reaction
mixture.
Acetonitrile is useful as the second diluent. The reaction mixture is
typically stirred for a
period of between about 2 and about 72 hours resulting in deprotection of
compound 8
and formation of the intermediate grade solid sulfate salt of compound 1,
which is
typically predominantly the crystalline hydrate of the sulfate of compound 1.
The
intermediate grade product can be isolated conventionally, for example by
filtration.
The hydrate form may be obtained by recrystallization of the intermediate
grade
sulfate product, for example, by suspending the intermediate grade product in
acetonitrile
with heating, adding water to promote dissolution, cooling to ambient
temperature, and
isolating the recrystallized hydrate form, as described in Example 2 below.
The crystalline sulfate salt of compound 1 may be obtained from the
intermediate
grade solid product of the deprotection step described above. The intermediate
grade
product is dispersed in an inert diluent comprising methanol at a
concentration between
about 5 and about 400 mg/mL, including between about 50 and about 200 mg/mL.
Surprisingly, it has been determined that a methanol and water combination,
having up to
% water, including between about 0 and about 15 % water, and between about 5
and
about 15 % water, is a useful diluent for preparing an anhydrous crystalline
salt. In
particular, a methanol and water combination comprising about 10 % water is
useful for
recrystallization of the intermediate grade product to the anhydrous sulfate
salt of the
20 present invention.
In a typical recrystallization process, the reaction mixture is heated until
complete
dissolution is obtained, for example the reaction mixture is heated to about
65 C, and
then cooled to between about 5 and about 22 C over a period of between about
2 and
about 24 hours. Optionally, seed crystals of the anhydrous sulfate salt can be
added when
25 the reaction mixture is below the dissolution temperature. The resulting
crystals are
recovered conventionally, for example, by filtration.
According to yet another process, the hydrate form may be prepared from the
crystalline sulfate (anhydrous) form. Typically, the crystalline sulfate is
first converted to
a more soluble, amorphous form, for example by lyophilization or rapid
evaporation of a
solution prepared from the crystalline sulfate. The amorphous solid sulfate
material is
then dispersed in an aqueous solvent system, for example 25 % water and 75 %
acetonitrile and optionally agitated for a time period, greater than about 12
or greater than
about 24 hours, at temperatures in the range of about 0 to about 65 C.
Typically, the
11

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temperature is first raised to about 65 C and then lowered to between about 5
and about
20 C. The resulting crystalline hydrate form is recovered conventionally.
Accordingly, in a method aspect, among other processes, the invention provides
a
process for preparing a crystalline sulfate salt of compound 1, the process
comprising:
(a) contacting a protected precursor to compound 1 in which the hydroxy groups
are
protected, with sulfuric acid to form a first reaction mixture; (b) isolating
an intermediate
grade solid sulfate salt of compound 1 from the first reaction mixture; (c)
dispersing the
intermediate grade solid sulfate salt in a diluent comprising methanol to form
a second
reaction mixture; and (d) isolating the crystalline sulfate salt from the
second reaction
mixture.
Further, in a composition aspect, the invention provides the N-
cyclohexylmethyl-
(2-oxoethyl)-carbamic acid benzyl ester bisulfite adduct 3 useful for
preparing
compound 1. As described in Preparation 1, the bisulfite adduct 3 can be
prepared by
reductive amination of cyclohexanecarboxaldehyde by 2,2-diethoxyethylamine
using
sodium triacetoxyborohydride, followed by addition of an amino-protecting
group,
deprotection of the aldehyde functionality, and conversion to the bisulfite
adduct by
reaction with sodium bisulfite. Alternatively, the initial reductive amination
can be
performed by catalytic hydrogenation. Suitable hydrogenation catalysts,
include, but are
TM
not limited to, palladium, platinum, and Raney nickel catalysts.
Pharmaceutical Compositions
The crystalline sulfate salt forms of the invention are typically administered
to a
patient in the form of a pharmaceutical composition or formulation. Such
pharmaceutical
compositions may be administered to the patient by any acceptable route of
administration
including, but not limited to, oral, rectal, vaginal, nasal, inhaled, topical
(including
transdermal) and parenteral modes of administration.
Accordingly, in one of its compositions aspects, the invention is directed to
a
pharmaceutical composition comprising a pharmaceutically-acceptable carrier or

excipient and a therapeutically effective amount of a crystalline sulfate salt
of
compound 1 or a solvate thereof. Optionally, such pharmaceutical compositions
may
contain other therapeutic and/or formulating agents if desired. When
discussing
compositions, it is understood the term "salt of the invention" includes the
crystalline
sulfate salt of compound 1 as well as solvates, in particular, the hydrate,
thereof.
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The pharmaceutical compositions of the invention typically contain a
therapeutically effective amount of the active agent, present in the form of a
salt of the
invention. Typically, such pharmaceutical compositions will contain from about
0.1 to
about 95 % by weight of the active agent; preferably, from about 5 to about 70
% by
weight; and more preferably from about 10 to about 60 % by weight of the
active agent.
Any conventional carrier or excipient may be used in the pharmaceutical
compositions of the invention. The choice of a particular carrier or
excipient, or
combinations of carriers or excipients, will depend on the mode of
administration being
used to treat a particular patient or type of medical condition or disease
state. In this
regard, the preparation of a suitable pharmaceutical composition for a
particular mode of
administration is well within the scope of those skilled in the pharmaceutical
arts.
Additionally, the carriers or excipients used in the pharmaceutical
compositions of this
invention are commercially-available. By way of further illustration,
conventional
formulation techniques are described in Remington: The Science and Practice of
Pharmacy, 20th Edition, Lippincott Williams & White, Baltimore, Maryland
(2000); and
H.C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th
Edition,
Lippincott Williams & White, Baltimore, Maryland (1999).
Representative examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, the following: sugars,
such as lactose,
glucose and sucrose; starches, such as corn starch and potato starch;
cellulose, such as
microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl
cellulose,
ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin;
talc; excipients,
such as cocoa butter and suppository waxes; oils, such as peanut oil,
cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such
as propylene
glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;
esters, such
as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;
Ringer's solution;
ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible
substances
employed in pharmaceutical compositions.
Pharmaceutical compositions are typically prepared by thoroughly and
intimately
mixing or blending the active agent with a pharmaceutically-acceptable carrier
and one or
more optional ingredients. The resulting uniformly blended mixture can then be
shaped
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or loaded into tablets, capsules, pills and the like using conventional
procedures and
equipment.
The pharmaceutical compositions of the invention are preferably packaged in a
unit dosage form. The term "unit dosage form" refers to a physically discrete
unit suitable
for dosing a patient, i.e., each unit containing a predetermined quantity of
active agent
calculated to produce the desired therapeutic effect either alone or in
combination with
one or more additional units. For example, such unit dosage forms may be
capsules,
tablets, pills, and the like, or unit packages suitable for parenteral
administration.
In one embodiment, the pharmaceutical compositions of the invention are
suitable
for oral administration. Suitable pharmaceutical compositions for oral
administration
may be in the form of capsules, tablets, pills, lozenges, cachets, dragees,
powders,
granules; or as a solution or a suspension in an aqueous or non-aqueous
liquid; or as an
oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and
the like; each
containing a predetermined amount of a compound of the present invention as an
active
ingredient.
When intended for oral administration in a solid dosage form (i.e., as
capsules,
tablets, pills and the like), the pharmaceutical compositions of the invention
will typically
comprise the active agent and one or more pharmaceutically-acceptable
carriers, such as
sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid
dosage
forms may also comprise: fillers or extenders, such as starches,
microcrystalline cellulose,
lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose
and/or acacia;
humectants, such as glycerol; disintegrating agents, such as agar-agar,
calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and/or sodium
carbonate; solution
retarding agents, such as paraffin; absorption accelerators, such as
quaternary ammonium
compounds; wetting agents, such as cetyl alcohol and/or glycerol monostearate;

absorbents, such as kaolin and/or bentonite clay; lubricants, such as talc,
calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or
mixtures
thereof; coloring agents; and buffering agents.
Release agents, wetting agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be present in the
pharmaceutical compositions of the invention. Examples of pharmaceutically-
acceptable
antioxidants include: water-soluble antioxidants, such as ascorbic acid,
cysteine
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hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite .and the
like; oil-
soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole,
butylated
hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and
metal-
chelating agents, such as citric acid, ethylenediamine tetraacetic acid,
sorbitol, tartaric
acid, phosphoric acid, and the like. Coating agents for tablets, capsules,
pills and like,
include those used for enteric coatings, such as cellulose acetate phthalate,
polyvinyl
acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid-
methacrylic
acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl
cellulose,
hydroxypropyl methyl cellulose acetate succinate, and the like.
Pharmaceutical compositions of the invention may also be formulated to provide
slow or controlled release of the active agent using, by way of example,
hydroxypropyl
methyl cellulose in varying proportions; or other polymer matrices, liposomes
and/or
microspheres. In addition, the pharmaceutical compositions of the invention
may
optionally contain opacifying agents and may be formulated so that they
release the active
ingredient only, or preferentially, in a certain portion of the
gastrointestinal tract,
optionally, in a delayed manner. Examples of embedding compositions which can
be
used include polymeric substances and waxes. The active agent can also be in
micro-
encapsulated form, if appropriate, with one or more of the above-described
excipients.
Suitable liquid dosage forms for oral administration include, by way of
illustration, pharmaceutically-acceptable emulsions, microemulsions,
solutions,
suspensions, syrups and elixirs. Liquid dosage forms typically comprise the
active agent
and an inert diluent, such as, for example, water or other solvents,
solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl
acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (esp.,
cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofiiryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Suspensions,
in addition to the active ingredient, may contain suspending agents such as,
for example,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,

microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth,
and mixtures thereof.
The salts of this invention can also be administered parenterally (e.g. by
intravenous, subcutaneous, intramuscular or intraperitoneal injection). For
parenteral
administration, the active agent is typically admixed with a suitable vehicle
for parenteral

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administration including, by way of example, sterile aqueous solutions,
saline, low
molecular weight alcohols such as propylene glycol, polyethylene glycol,
vegetable oils,
gelatin, fatty acid esters such as ethyl oleate, and the like. Parenteral
formulations may
also contain one or more anti-oxidants, solubilizers, stabilizers,
preservatives, wetting
agents, emulsifiers, or dispersing agents. These formulations may be rendered
sterile by
use of a sterile injectable medium, a sterilizing agent, filtration,
irradiation, or heat.
Alternatively, the pharmaceutical compositions of the invention are formulated
for
administration by inhalation. Suitable pharmaceutical compositions for
administration by
inhalation will typically be in the form of an aerosol or a powder. Such
compositions are
generally administered using well-known delivery devices, such as a metered-
dose
inhaler, a dry powder inhaler, a nebulizer or a similar delivery device.
When administered by inhalation using a pressurized container, the
pharmaceutical compositions of the invention will typically comprise the
active ingredient
and a suitable propellant, such as dichlorodifluoromethane,
trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Additionally,
the
pharmaceutical composition may be in the form of a capsule or cartridge (made,
for
example, from gelatin) comprising a compound of the invention and a powder
suitable for
use in a powder inhaler. Suitable powder bases include, by way of example,
lactose or
starch.
The salts of the invention can also be administered transdermally using known
transdermal delivery systems and excipients. For example, the active agent can
be
admixed with permeation enhancers, such as propylene glycol, polyethylene
glycol
monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch
or similar
delivery system. Additional excipients including gelling agents, emulsifiers
and buffers,
may be used in such transdermal compositions if desired.
If desired, the salts of this invention may be administered in combination
with one
or more other therapeutic agents. In this embodiment, a salt of this invention
is either
physically mixed with the other therapeutic agent to form a composition
containing both
agents; or each agent is present in separate and distinct compositions which
are
administered to the patient simultaneously or sequentially.
For example, a salt of the invention can be combined with second therapeutic
agent using conventional procedures and equipment to form a composition
comprising a
compound 1 and a second therapeutic agent. Additionally, the therapeutic
agents may be
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combined with a pharmaceutically acceptable carrier to form a pharmaceutical
composition comprising a salt of the invention, a second therapeutic agent and
a
pharmaceutically acceptable carrier. In this embodiment, the components of the

composition are typically mixed or blended to create a physical mixture. The
physical
mixture is then administered in a therapeutically effective amount using any
of the routes
described herein. Alternatively, the therapeutic agents may remain separate
and distinct
before administration to the patient. In this embodiment, the agents are not
physically
mixed together before administration but are administered simultaneously or at
separate
times as separate compositions. Such compositions can be packaged separately
or may be
packaged together as a kit. The two therapeutic agents in the kit may be
administered by
the same route of administration or by different routes of administration.
Any therapeutic agent compatible with the present active agent may be used as
the
second therapeutic agent. In particular, prokinetic agents acting via
mechanisms other
than mu opioid receptor antagonism may be used in combination with the present
compounds. For example, 5-HT4 receptor agonists, such as tegaserod,
renzapride,
mosapride, prucalopride, 1-isopropy1-1H-indazole-3-carboxylic acid {(1S,3R,5R)-
8-[2-(4-
acetylpiperazin-l-yl)ethyl]-8-azabicyclo[3.2.1]oct-3-y1}amide , 1-isopropy1-2-
oxo-1,2-
dihydroquinoline-3-carboxylic acid {(1S,3R,5R)-8-[(R)-2-hydroxy-3-
(methanesulfonyl-
methyl-amino)propy1]-8-azabicyclo[3.2.1]oct-3-y1) amide, or 4-(4- {[(2-
isopropy1-1H-
b enzoimidazole-4-carb onyl)amino]methyll-pip eridin-l-ylmethyl)pip eridine-l-
carboxylic
acid methyl ester may be used as the second therapeutic agent.
Additional useful prokinetic agents include, but are not limited to, 5-HT3
receptor
agonists (e.g. pumosetrag), 5-HTIA receptor antagonists (e.g. AGI 001), alpha-
2-delta
ligands (e.g. PD-217014), chloride channel openers (e.g. lubiprostone),
dopamine
antagonists (e.g. itopride, metaclopramide, domperidone), GABA-B agonists
(e.g.
baclofen, AGI 006), kappa opioid agonists (e.g. asimadoline), muscarinic M1
and M2
antagonists (e.g. acotiamide), motilin agonists (e.g. mitemcinal), guanylate
cyclase
activators (e.g. MD-1100) and ghrelin agonists (e.g. Tzp 101, RC 1139).
In addition, the salts of the invention can be combined with opioid
therapeutic
agents. Such opioid agents include, but are not limited to, morphine,
pethidine, codeine,
dihydrocodeine, oxycontin, oxycodone, hydrocodone, sufentanil, fentanyl,
remifentanil,
buprenorphine, methadone, and heroin.
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Numerous additional examples of such therapeutic agents are known in the art
and
any such known therapeutic agents may be employed in combination with the
compounds
of this invention. Secondary agent(s), when included, are present in a
therapeutically
effective amount, i.e. in any amount that produces a therapeutically
beneficial effect when
co-administered with a compound of the invention. Suitable doses for the other
therapeutic agents administered in combination with a compound of the
invention are
typically in the range of about 0.05 fig/day to about 100 mg/day.
Accordingly, the pharmaceutical compositions of the invention optionally
include
a second therapeutic agent as described above.
The following examples illustrate representative pharmaceutical compositions
of
the present invention:
Formulation Example A: Hard Gelatin Capsules for Oral Administration
A salt of the invention (50 g), spray-dried lactose (200 g) and magnesium
stearate
(10 g) are thoroughly blended. The resulting composition is loaded into a hard
gelatin
capsule (260 mg of composition per capsule).
Formulation Example B: Hard Gelatin Capsules for Oral Administration
A salt of the invention (20 mg), starch (89 mg), microcrystalline cellulose
(89 mg),
and magnesium stearate (2 mg) are thoroughly blended and then passed through a
No. 45
mesh U.S. sieve. The resulting composition is loaded into a hard gelatin
capsule (200 mg
of composition per capsule).
Formulation Example C: Gelatin Capsules for Oral Administration
A salt of the invention (10 mg), polyoxyethylene sorbitan monooleate (50 mg),
and starch powder (250 mg) are thoroughly blended and then loaded into a
gelatin
capsule (310 mg of composition per capsule).
Formulation Example D: Tablets for Oral Administration
A salt of the invention (5 mg), starch (50 mg), and microscrystalline
cellulose
(35 mg) are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. A
solution
of polyvinylpyrrolidone (10 wt% in water, 4 mg) is mixed with the resulting
powders, and
this mixture is then passed through a No. 14 mesh U.S. sieve. The granules so
produced
are dried at 50-60 C and passed through a No. 18 mesh U.S. sieve. Sodium
carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg) and talc (1 mg),
which have
previously been passed through a No. 60 mesh U.S. sieve, are then added to the
granules.
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After mixing, the mixture is compressed on a tablet machine to afford a tablet
weighing
100 mg.
Formulation Example E: Tablets for Oral Administration
A salt of the invention (25 mg), microcrystalline cellulose (400 mg), fumed
silicon
dioxide (10 mg), and stearic acid (5 mg) are thoroughly blended and then
compressed to
form tablets (440 mg of composition per tablet).
Formulation Example F: Single-scored Tablets For Oral Administration
A salt of the invention (15 mg), cornstarch (50 mg), croscarmellose sodium (25

mg), lactose (120 mg), and magnesium stearate (5 mg) are thoroughly blended
and then
compressed to form single-scored tablet (215 mg of compositions per tablet).
Formulation Example G: Suspension for Oral Administration
The following ingredients are thoroughly mixed to form a suspension for oral
administration containing 100 mg of active ingredient per 10 mL of suspension:
Ingredients Amount
Salt of the invention 0.1 g
Fumaric acid 0.5 g
Sodium chloride 2.0 g
Methyl paraben 0.15 g
Propyl paraben 0.05 g
Granulated sugar 25.5 g
Sorbito (70% solution) 12.85 g
Veegurrik (Vanderbilt Co.) 1.0 g
Flavoring 0.035 mL
Colorings 0.5 mg
Distilled water q.s. to 100 mL
Formulation Example H: Dry Powder Composition
A micronized salt of the invention (1 mg) is blended with lactose (25 mg) and
then
loaded into a gelatin inhalation cartridge. The contents of the cartridge are
administered
using a powder inhaler.
Formulation Example J: Injectable Formulation
A salt of the invention (0.1 g) is blended with 0.1 M sodium citrate buffer
solution
(15 mL). The pH of the resulting solution is adjusted to pH 6 using 1 N
aqueous
hydrochloric acid or 1 N aqueous sodium hydroxide. Sterile normal saline in
citrate
buffer is then added to provide a total volume of 20 mL.
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It will be understood that any form of salt of the invention, (i.e.
crystalline salt, or
solvate) that is suitable for the particular mode of administration, can be
used in the
pharmaceutical compositions discussed above.
Utility
The present active agent, 3 -endo-(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-
propionyl)amino]ethyll-8-aza-bicyclo[3.2.1]oct-3-yObenzamide sulfate, is an
antagonist
at the mu opioid receptor and therefore the salts of the invention are
expected to be useful
for treating medical conditions mediated by mu opioid receptors or associated
with mu
opioid receptor activity, i.e. medical conditions which are ameliorated by
treatment with a
mu opioid receptor antagonist. In particular, the salts of the invention are
expected to be
useful for treating adverse effects associated with use of opioid analgesics,
i.e. symptoms
such as constipation, decreased gastric emptying, abdominal pain, bloating,
nausea, and
gastroesophageal reflux, termed collectively opioid-induced bowel dysfunction.
The
present salt forms are also expected to be useful for treating post-operative
ileus, a
disorder of reduced motility of the gastrointestinal tract that occurs after
abdominal or
other surgery. In addition, it has been suggested that mu opioid receptor
antagonist
compounds, such as compound 1 may be used for reversing opioid-induced nausea
and
vomiting.
Since compound 1 has been shown to increase motility of the gastrointestinal
(GI)
tract in animal models, the salts of the invention are expected to be useful
for treating
disorders of the GI tract caused by reduced motility in mammals, including
humans. Such
GI motility disorders include, by way of illustration, chronic constipation,
constipation-
predominant irritable bowel syndrome (C-IBS), diabetic and idiopathic
gastroparesis, and
functional dyspepsia.
In one aspect, therefore, the invention provides a method of increasing
motility of
the gastrointestinal tract in a mammal, the method comprising administering to
the
mammal a therapeutically effective amount of a pharmaceutical composition
comprising a
pharmaceutically-acceptable carrier and a salt of the invention.
When used to treat disorders of reduced motility of the GI tract or other
conditions
mediated by mu opioid receptors, the salts of the invention will typically be
administered
orally in a single daily dose or in multiple doses per day, although other
forms of
administration may be used. For example, particularly when used to treat post-
operative

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ileus, the compounds of the invention may be administered parenterally. The
amount of
active agent administered per dose or the total amount administered per day
will typically
be determined by a physician, in the light of the relevant circumstances,
including the
condition to be treated, the chosen route of administration, the actual
compound
administered and its relative activity, the age, weight, and response of the
individual
patient, the severity of the patient's symptoms, and the like.
Suitable doses for treating disorders of reduced motility of the GI tract or
other
disorders mediated by mu opioid receptors will range from about 0.0007 to
about
20 mg/kg/day of active agent, including from about 0.0007 to about 1.4
mg/kg/day. For
an average 70 kg human, this would amount to from about 0.05 to about 100 mg
per day
of active agent.
In one aspect of the invention, the compounds of the invention are used to
treat
opioid-induced bowel dysfunction. When used to treat opioid-induced bowel
dysfunction, the compounds of the invention will typically be administered
orally in a
single daily dose or in multiple doses per day. Preferably, the dose for
treating opioid-
induced bowel dysfunction will range from about 0.05 to about 100 mg per day.
In another aspect of the invention, the compounds of the invention are used to
treat
post-operative ileus. When used to treat post-operative ileus, the compounds
of the
invention will typically be administered orally or intravenously in a single
daily dose or in
multiple doses per day. Preferably, the dose for treating post-operative ileus
will range
from about 0.05 to about 100 mg per day.
The invention also provides a method of treating a mammal having a disease or
condition associated with mu opioid receptor activity, the method comprising
administering to the mammal a therapeutically effective amount of a compound
of the
invention or of a pharmaceutical composition comprising a compound of the
invention.
The present active agent is optionally administered in combination with
another
therapeutic agent or agents, in particular, in combination with prokinetic
agents acting via
non-mu opioid mechanisms. Accordingly, in another aspect, the methods and
compositions of the invention further comprise a therapeutically effective
amount of
another prokinetic agent.
As described above, salts of the invention are mu opioid receptor antagonists.
The
invention further provides, therefore, a method of antagonizing a mu opioid
receptor in a
mammal, the method comprising administering a salt of the invention to the
mammal.
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Among other properties, the present active agent in freebase and sulfate salt
form
has been found to exhibit potent binding to mu opioid receptors and little or
no agonism
in mu receptor functional assays. Therefore, the salts of the invention are
potent mu
opioid receptor antagonists. Further, active agent has demonstrated
predominantly
peripheral activity as compared with central nervous system activity in animal
models.
Therefore, the salts of the invention can be expected to reverse opioid-
induced reductions
in GI motility without interfering with the beneficial central effects of
analgesia. These
properties, as well as the utility of the compounds of the invention, can be
demonstrated
using various in vitro and in vivo assays well-known to those skilled in the
art.
Representative assays are described in further detail in the following
examples.
EXAMPLES
The following synthetic and biological examples are offered to illustrate the
invention, and are not to be construed in any way as limiting the scope of the
invention.
In the examples below, the following abbreviations have the following meanings
unless
otherwise indicated. Abbreviations not defined below have their generally
accepted
meanings.
D1PEA = N,N-diisopropylethylamine
DMF = N,N-dimethylformamide
Et0Ac = ethyl acetate
Et0H = ethanol
MeTHF = 2-methyltetrahydrofuran
MTBE = tert-butyl methyl ether
NaHMDS = sodium bis(trimethylsilyl)amide
PyBop = benzotriazol-1-yloxytripyrrolidino-
phosphonium hexafluorophosphate
psi = pounds per square inch
Rt retention time
THF = tetrahydrofuran
Reagents and solvents were purchased from commercial suppliers (Aldrich,
Fluka,
Sigma, etc.), and used without further purification. Reactions were run under
nitrogen
atmosphere, unless noted otherwise. Progress of reaction mixtures was
monitored by thin
layer chromatography (TLC), analytical high performance liquid chromatography
(anal.
HPLC), and mass spectrometry. Endo/exo ratios of products were determined by
HPLC
analysis using the protocols described below. Reaction mixtures were worked up
as
22

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described specifically in each reaction; commonly they were purified by
extraction and
other purification methods such as temperature-, and solvent-dependent
crystallization,
and precipitation. Characterization of reaction products was routinely carried
out by mass
and 111-NMR spectrometry. For NMR measurement, samples were dissolved in
deuterated solvent (CD30D, CDC13, oi.DMSO-d6), and 11-I-NMR spectra were
acquired
TM
with a Varian Gemini 2000 instrument (300 MHz) under standard observation
conditions.
Mass spectrometric identification of compounds was performed by an
electrospray
TM
ionization method (ESMS) with an Applied Biosystems (Foster City, CA) model
API 150
TM
EX instrument or an Agilent (Palo Alto, CA) model 1100 LC/MSD instrument.
General HPLC Conditions
TM
Column: Zorbax SB-Aq, 5 gm. 4.6 x 250 mm
Column temperature: 40 C
Flow rate: 1.0 mL/min
Mobile Phases: A = Water/ACN (98:2) + 0.1 % TFA
B = Water/ACN (10:90) + 0.1 % TFA,
Injection volume: 10 iL
Detector wavelength: 214 nm
HPLC method 1
Crude compounds were dissolved in Water/ACN (50:50) at about 1 mg/mL and
analyzed using the following gradient over 20 min (time (min)/ % B): 0/10,
2.5/20, 9/75,
15/90, 17/90, 18/10, 20/10.
HPLC method 2
Compounds were dissolved in Water/ACN (90:10) at about 1 mg/mL and
analyzed using the following gradient over 30 min (time (min)/ % B): 0/10,
13/10, 23/65,
28/90, 29/90, 30/10.
HPLC method 3
Compounds were dissolved in Water/ACN (90:10) at about 1 mg/mL and
analyzed using the following gradient over 55 min (time (min)/ % B): 0/10,
10/20, 46/75,
47/90, 50/10, 55/10.
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Preparation 1: Synthesis of N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid
benzyl ester bisulfite adduct
a. Preparation of N-cyclohexylmethyl-(2,2-diethoxyethyl)amine
To a mixture of 2,2-diethoxyethylamine (209 mL, 1.43 mol) and MeTHF (1050 L)
was added cyclohexanecarbaldehyde (107 mL, 0.89 mol). The reaction mixture was
stirred for 30 mm at room temperature and cooled to 0 C. Sodium
triacetoxyborohydride
(378 g, 1.79 mol) was added over 40 min and the reaction mixture was stirred
for 2 h and
cooled to 0 C. 1 M NaOH (1 L) was added. The organic layer was washed with
brine in
water (1:1,2 x 1 L) and the volume was reduced to ¨ 20%. MeTHF (1 L) was added
and
the volume reduced to ¨ 20 %. The solution of the crude title intermediate was
used
directly in the next step.
b. Preparation of N-cyclohexylmethyl-(2,2-diethoxyethyl)carbamic acid benzyl
ester
To the product of the previous step (-213 g, ¨0.9 mol) was added MeTHF (2 L)
and DIPEA (233 mL, 1.34 mol). The reaction mixture was cooled to 0 C and
benzylchloroformate (140 mL, 0.98 mol) was added dropwise. The reaction
mixture was
stirred for 30 min at 0 C, for 2 h at 0 C to room temperature, and then for
1 h at room
temperature. Water (1.6 L) was added and the reaction mixture was stirred for
10 min.
The phases were separated and the organic layer was washed with sodium
bicarbonate
(1.6 L) and water (1.6 L). The layers were separated and the organic layer was
reduced to
about 20 %. MeTHF (1 L) was added and the volume reduced to ¨ 20 %. The
solution of
the crude title intermediate was used directly in the next step.
c. Synthesis of N-cyclohexvimethyl-(2-oxoethyl)-carbamic acid benzyl ester
bisulfite
adduct
To the product of the previous step (-302 g, ¨0.62 mol) and acetonitrile (2 L)
was
added 1 M HC1 (2 L) and the reaction mixture was stirred at 30 C for 7 h.
Ethyl acetate
(2 L) was added and the reaction mixture was stirred for 10 min. The phases
were
separated, the organic layer was washed with 1 M HC1 (1.5 L), the phases were
again
separated and the organic layer was washed with 0.5 M HC1 (1 L). Sodium
bisulfite
(71.4 g, 0.69 mol) was added and the reaction mixture was stirred overnight,
and then
filtered. The reactor and filter cake were washed with ethyl acetate (1 L).
The resulting
solution was dried in air for 2 h and under vacuum overnight to provide the
title
compound as a white solid (199 g, >99 % area purity by HPLC). The filtrate was
treated
by the same procedure to provide a second lot of the title compound (30 g).
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Preparation 2: Synthesis of 3-endo-(8-azabicyclo[3.2.11oct-3-y1)-benzamide
a. Preparation of 8-benzy1-3-exo-(3-methoxypheny1)-8-azabicyclo[3.2.1]octan-3-
ol
To a 3L flask was added cerous chloride powder (194 g, 0.79 mol). The flask
was
flushed with nitrogen and THF (800 mL) was added. The reaction mixture was
stirred at
25 C for lh. To the mixture was added ¨1M 3-methoxyphenyl magnesium bromide
in
THF (800 mL, 0.87 mol) dropwise. The resulting slurry was stirred at 3 C for
1.5 hours.
A solution of 8-benzy1-8-aza-bicyclo[3.2.1]octan-3-one (120.4 g, 0.56 mol) in
THF (200
mL) was then added dropwise, while maintaining the internal temperature at -5
C. The
resulting solution was stirred for 15 min. The reaction mixture was added to a
flask
containing 6 N HC1 (800 mL) maintaining the temperature at 10 C. After
solvent was
removed by rotary evaporation, the reaction mixture was stirred at room
temperature
overnight. The solids were isolated by filtration, washed with 6N HCL (70 mL)
and
acetonitrile (3 x 70 mL), and dried to provide the HC1 salt of the title
intermediate as an
off-white solid (161 g).
b. Preparation of 8-benzy1-3-(3-methoxypheny1)-8-azabicyclop.2.1]oct-2-ene
To a 3 L flask was added 8-benzy1-3-exo-(3-methoxypheny1)-8-
azabicyclo[3.2.1]octan-3-ol hydrochloride (383.9 g, 1.06 mol), 6 M HC1 (800
mL), and
MeTHF (200 mL). The resulting slurry was heated at 70 C for 2.5 h under
nitrogen. The
reaction mixture was transferred to a 12 L reactor and cooled to 10 C. The
reaction flask
was washed with MeTHF (1 L) that was added to the 12 L reactor. NaOH (50 wt %
in
water, 200 mL) was added and additional NaOH (50 wt %, 150 mL) was added in
portions until pH ¨13 was reached. The phases were separated, the water layer
was
extracted with MeTHF (1 L), and combined MeTHF layers were washed with brine
(1 L).
Solvent was reduced by rotary evaporation at 30 to 40 C yielding the title
intermediate
(360 g) as a thick oil. Et0H (1.5 L) was added and the volume was reduced to ¨
500 mL
and then adjusted to 1.8 L.
c. Preparation of 3-endo-(3-methoxypheny1)-8-azabicyclo[3.2.1]octane
To 8-benzy1-3-(3-methoxypheny1)-8-azabicyclo[3.2.1]oct-2-ene (in Et0H 95 %,
400 mL, 0.20 mol), prepared in the previous step, was added 6 M HC1 (45 mL)
and then
MeTHF (50 mL). The reaction mixture was purged with nitrogen, heated to 40 C
and
palladium on carbon (10 weight %, 8 g) was added. The reactor was pressurized
with
hydrogen (3 x 20 psi) and then hydrogenated at 20 psi at 40 C for 18 h. The
reaction

CA 02678073 2014-05-13
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TM
mixture was filtered through Celite, concentrated, washed with MeTHF (2 x 100
mL),
filtered through a coarse glass filter, washed with MeTHF (10 mL) and dried on
the filter
to provide the HC1 salt of the title intermediate as white solid (31 g, single
isomer, (exo
isomer undetectable by HPLC)). An additional 5.2 g of product was recovered
from the
mother liquor.
d. Preparation of 3-en do-(8-azabicyclo[3.2.1]oct-3-y1)-phenol
To a 500 mL flask was added 3-endo-(3-methoxypheny1)-8-
azabicyclo[3.2.1]octane hydrochloride (115 g, 0.45 mol) and hydrobromic acid
(48 weight
% in water, 100 mL, 0.88 mol). The mixture was heated to 120 C and held at
that
temperature for 24 h with stirring. Additional hydrobromic acid solution (25
mL) was
added and the reaction mixture was heated with stirring for 6 h and then
cooled to 70 C.
Acetonitrile (200 mL) was added and the resulting slurry was cooled to 10 C
and then
filtered, and the filter cake was washed with acetonitrile (50 mL) to yield
the HBr salt of
the title intermediate (99 g, > 99 % pure) as a white granular solid.
e. Preparation of 2,2,2-trifluoro-143-endo-(3-hydroxypheriy1)-8-
azabicyclo[3.2.11oct-8-
vliethanone
To a solution of 3-en do-(8-azabicyclo[3.2.1]oct-3-y1)-phenol hydrobromide
(54.4 g, 0.19 mol), toluene (210 mL), and triethylamine (40 mL, 0.29 mol), was
added
trifluoroacetic anhydride (54 mL, 0.38 mol) over 20 min. The reaction mixture
was
stirred at 40 C for 2 h. Ethyl acetate (370 mL) and brine in water (1:1, 265
mL) were
added. The reaction mixture was stirred for 15 min, the phases were separated.
To the
organic layer was added saturated sodium bicarbonate (300 mL) and the mixture
was
stirred vigorously overnight. The phases were separated and the organic layer
was
washed with brine in water (1:1, 265 mL) dried over sodium sulfate and most of
the
solvent was removed by rotary evaporation. Toluene (100 mL) was added and the
solvent
removed by rotary evaporation to provide the crude title intermediate.
f. Preparation of trifluoromethanesulfonic acid 3-endo-j8-(2,2,2-trifluoro-
aceq4)-8-
azabicyclo[3.2.1]oct-3-yliphenyl ester
To a 500 mL flask was added the ethyl acetate solution (220 mL) of the
intermediate of the previous step (32.8 g, 0.11 mol) and triethylamine (23 mL.
0.17 mol).
The solution was cooled to 5 C and trifluoromethane sulfonyl chloride (14 mL,
0.13 mol)
was added dropwise. The mixture was allowed to warm to 25 C and stirred at
that
temperature for 1 h. Saturated sodium bicarbonate (200 mL) was added, the
layers were
26

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separated, brine (150 mL) was added to the organic layer, the layers were
again separated,
and solvent was removed from the organic layer to provide the crude title
intermediate.
g. Preparation of 3-endo-[8-(2,2,2-trifluoroacety1)-8-azabicyclo[3.2.1joct-3-
y1J-
benzonitrile
To a 100 mL flask was added trifluoromethanesulfonic acid 3-endo48-(2,2,2-
trifluoro-acety1)-8-azabicyclo[3.2.11oct-3-yl]phenyl ester (25.3 g, 58.7
mmol),
tris(dibenzylideneacetone) dipalladium (0) (0.81g, 0.9 mmol), 1,1'-
bis(diphenylphosphino) ferrocene (1.01 g, 1.8 mmol), and zinc cyanide (4.2 g,
35.8 mmol). Three times, the flask was purged with nitrogen for 5 min and then
placed
under house vacuum for 5 min. To the flask was added DMF (150 mL) and
distilled
water (2.5 mL). The solution was purged with nitrogen with stirring for 10
min, heated to
120 C and stirred at 120 C under nitrogen for 4 h. When the reaction was
completed
g of product from a previous lot, prepared by the same procedure, was added
and
stirred for 20 min.
15 Most of the solvent was removed by distillation and the solution was
cooled to
22 C. To the solution was added ethyl acetate (445 mL) and the resulting
solution was
filtered through Celite. Sodium bicarbonate (450 mL) was added and the
solution was
stirred for 15 min. The layers were separated and the organic layer was washed
with
diluted brine (2 x 95 mL), and filtered through sodium sulfate. The volume was
reduced
20 to about 50 mL by removal of ethyl acetate. Isopropyl alcohol (150 mL)
was added and
the solution was agitated at 22 C for 1 h. Solids were isolated by filtration
and washed
with isopropyl alcohol (2 x 25 mL) to provide the title intermediate (33.5 g,
100 % pure
by HPLC) as an off-white/light brown solid. A second crop of product (6.3 g, >
98 %
pure by HPLC) was isolated from the filtrate.
h. Synthesis of 3-endo-(8-azabicyclo[3.2.1]oct-3-y1)-benzamide
A solution of 3-endo-[8-(2,2,2-trifluoroacety1)-8-azabicyclo[3.2.1]oct-3-y1]-
benzonitrile (10 g, 32 mmol) in sulfuric acid (96 %, 12 mL) was heated to 50
C with
stirring and held at that temperature with stirring for 2 h. The reaction
mixture was
cooled to 22 C and added slowly to a 500 mL flask containing 5 N NaOH (90 mL)
and
methanol (100 mL) which was cooled to 10 C. Salt precipitates were filtered
and the
filtrate was stirred at 22 C for 1 h. The reaction mixture was concentrated
under reduced
pressure. To the residue was added MeTHF (150 mL) and the reaction mixture was

stirred at 22 C for 5 min. The layers were separated and MeTHF (100 mL) was
added to
27

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the aqueous layer. The layers were separated and brine (150 mL) was added to
the
combined organic layers. The layers were separated and the organic layer was
dried over
potassium carbonate and filtered, and the solvent was removed. A mixture of
Et0H
(25 mL) and concentrated HC1 (2.6 mL) was added to the residue with stirring
and then
MTBE (25 mL) was added and the solution was stirred at 22 C. Precipitated
solids were
filtered and air dried to provide the HC1 salt of the title compound (8 g, 97
% purity by
HPLC) as a white solid.
Preparation 3: Synthesis of 3-endo-(8-azabicyclo[3.2.11oct-3-yl)benzamide
a. Preparation of trifluoro-methanesulfonic acid 8-benzy1-8-
azabicyclo[3.2.1]oct-2-en-3-
yl ester
To a 500 mL flask was added 8-benzy1-8-azabicyclo[3.2.1]octan-3-one
hydrochloride (50.4 g, 200 mmol), Et0Ac (160 mL), and 4 N NaOH (50 mL). The
reaction mixture was heated to 30 C and stirred at that temperature for 1 h.
The layers
were separated and the aqueous layer was discarded. The volume of the organic
layer was
reduced to ¨40 mL by rotary evaporation and THF (270 mL) was added.
The resulting solution was added to a 1L flask and cooled to -20 C. A NaHMDS
solution (1 M in THF, 230 mL, 230 mmol) was added to the flask over 15 min.
The
reaction mixture was stirred at -20 5 C for 1 h. N-phenyl-
bis(trifluoromethanesulfonimide (82.2 g, 230 mmol) was added to the reaction
mixture in
portions over 5 min and the mixture was stirred at ¨ 20 C to -10 C for 1 h.
To the
reaction mixture was added 1 N NaOH (200 mL) and the mixture was allowed to
warm to
22 C with stirring. Solvent was partially removed by rotary evaporation at 30
C to a
volume of 450 mL. To the remaining reaction mixture was added Et0Ac (300 mL)
and
heptane (150 mL). The mixture was stirred at 22 C for 5 mm. The layers were
separated
and the aqueous layer was discarded. The organic layer was washed with 1N NaOH
(3 x
450 mL). The aqueous layers were discarded. The organic layer was concentrated
by
rotary evaporation to provide the title intermediate (77 g, >96 % purity by
HPLC
method 1).
NMR (d6-DMSO, 400 MHz): 5 (ppm) 7.25-7.35 (m, 5H), 6.05 (d, J=5.2, 1H),
3.64 (q, J=13.2, 2H), 3.40-3.44 (m, 2H), 2.77 (d, J=16.4, 1H), 1.79-2.09 (m,
5H), 1.52-
1.59 (m, 1H).
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b. Preparation of 3-(8-benzy1-8-azabicyclor3.2.11oct-2-en-3-yl)benzamide
To the crude product of the previous step was added THF (420 mL) and the
solution was purged with nitrogen for 5 min. To a 2 L flask was added
3-carbamoylphenyl boronic acid (98 %, 33.0 g, 200 mmol), palladium (II)
acetate (98 %.
0.46 g, 2 mmol), 1,1'-bis(diphenylphosphino)ferrocene (97 %, 1.1 g, 2 mmol).
and
potassium fluoride (34.9 g, 600 mmol) followed by the THF solution of
trifluoro-
methanesulfonic acid 8-benzy1-8-aza-\bicyclo[3.2.1]oct-2-en-3-y1 ester. The
resulting
mixture was purged with nitrogen for 5 min, heated to reflux (67 C) under
nitrogen and
stirred for 2 h. The reaction mixture was cooled to 30 C, then Et0Ac (500 mL)
and
1 N NaOH (500 mL) were added and the mixture was stirred at 22 C for 10 min.
The
layers were separated and the aqueous layer was discarded. The organic layer
was washed
with a mixture of brine (250 mL) and water (250 mL) and stirred for 5 min. The
layers
were separated and the aqueous layer was discarded. The organic layer was
briefly dried
over Na2SO4, filtered, and solvent was partially removed. Product precipitated
as light
yellow solids during solvent removal. The resulting slurry (about 200 mL) was
filtered
and the solids were washed with cold Et0Ac (0 C, 100 mL) and dried under high

vacuum at 25 C to provide the title intermediate (42.5 g) as a light yellow
solid.
The mother liquor and the above washes were combined and concentrated and the
resulting slurry (about 100 mL) was stirred at 5 C for 30 min and filtered.
The filtered
solids were washed with cold Et0Ac (0 C, 30 mL) and dried under high vacuum
to
provide a second crop of the title intermediate (7 g, combined yield 78 %, >
98.5 % pure
by HPLC method 1).
(m/z): [M+H] paled for C211122N20, 319.18; found 319.4. II-1 NMR (CDC13, 400
MHz): ö (ppm) 7.9 (s, 1H), 7.63 (d, J=6.4, 1H), 7.57 (d, J=6.4, 1H), 7.21-7.42
(m, 6H),
6.38 (d, J=4.4, 1H), 6.13 (s, br, 1H), 5.83 (s, br, 1H), 3.68-3.76 (m, 2H),
3.46-3.51 (m,
2H), 2.92 (d, J=17.2, 1H), 2.18-2.26 (m, 1H), 2.04-2.12 (m, 2H), 1.86-1.92 (m,
1H), 1.58-
1.65 (m, 1H).
c. Synthesis of 3-endo-(8-azabicyclo[3.2.1]oct-3-yl)benzamide
To a 1 L hydrogenation vessel was added 3-(8-benzy1-8-azabicyclo[3.2.1]oct-2-
en-
3-yl)benzamide (40 g, 125 mmol), Et0H (800 mL), 6 M HC1 (42 mL) and water (80
mL)
and the mixture was stirred at 22 C until complete dissolution was observed.
The
reaction mixture was purged with nitrogen for 5 min while being heated to 30
C over
5 min. To the mixture was added 10 wt % Pd/C (50 % in water, 4 g). The mixture
was
29

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purged at atmospheric pressure with hydrogen for 5-10 min while being heated.
The
mixture was stirred at 50 C under a flow of hydrogen at < 5 psi (<0.34
atmospheres) for
h, resulting in >99 % conversion of the reactants, according to HPLC analysis.
The
solution was cooled to 30 C and filtered through Celite to provide a solution
of the crude
5 HC1 salt of the title compound with an endo:exo ratio of'-93:7 by HPLC
method 2 endo
Rt=10.97, exo Rt=12.67. (m/z): [M+H] calcd for C14H18N20, 231.15; found 231.2.

Water was removed from the crude product by azeotropic distillation at 30 C
in
Et0H (-80 mL) to provide a slurry that was heated to 60 C until complete
dissolution.
The solution was cooled to 35 C and seed crystals of the product (0.05 g)
were added.
(The seed crystals were prepared according to the process described in
Preparation 2.)
The resulting slurry was stirred at 22 C for 30 min, MTBE (120 mL) was added
slowly,
and the slurry was stirred at 22 C for 4 h and then at 0 C for 1 h. The
resulting solids
were filtered, washed with cold Et0H and dried under high vacuum to provide
the HC1
salt of the title compound (24.5 g) as a white powder (75 % yield, >98.5 %
purity < 0.4 %
exo isomer by HPLC method 3, endo Rt=8.67, exo Rt=9.43).
(m/z): [M+H] calcd for C14H18N20 231.15; found 231.2. 1H NMR (d6-DMSO,
400 MHz): 8 (ppm) 9.13 (s, br, 1H), 9.03 (s, br, 1H), 8.05 (s, 1H), 7.93 (s,
1H), 7.73 (d,
J=7.6, 114), 7.58 (d, J=7.6, 1H), 7.40 (t, J=7.6, 2H), 3.97 (s, 2H), 3.17-3.23
(m, 1H), 2.39-
2.46 (m, 2H), 2.19-2.24 (m, 2H), 1.86-1.89 (m, 2H), 1.59-1.63 (m, 2H).
Example 1A: Synthesis of crystalline 3-endo-(8-{2-[cyclohexylmethyl-((S)-
2,3-dihydroxy-propionyl)aminolethyl}-8-aza-bicyclo[3.2.11oct-3-y1)benzamide
sulfate
a. Preparation of N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid benzyl ester
To a 100 mL flask was added N-cyclohexylmethyl-(2-oxoethyl)-carbamic acid
benzyl ester bisulfite adduct (3.94 g, 1 mmol) and MeTHF (35 mL), followed by
water
(25 mL). The resulting slurry was stirred at room temperature for 5 min and 1
M NaOH
(8 mL) was added. The reaction mixture was stirred at room temperature for 45
min. The
layers were separated and the volume of the organic layer was reduced to ¨8 mL
to
provide the crude title intermediate.
b. Preparation of 243-endo-(3-carbamoylpheny1)-8-azabicyclo[3.2.1]oct-8-yll-
ethyl}cyclohexylmethyl-carbamic acid benzyl ester
To the product of the previous step was added DMF (15 mL) followed by 3-endo-
(8-azabicyclo[3.2.1]oct-3-y1)-benzamide hydrochloride (2.67 g, 1 mmol) and
then DMF

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(10 mL). The mixture was stirred at room temperature for 30 min, cooled to 10
C and
then sodium triacetoxyborohydride (4.25 g, 2 mmol) was added. The reaction
mixture
was stirred at room temperature for 90 min and then cooled to 10 C. Isopropyl
acetate
(100 mL) was added, followed by 1 M NaOH (50 mL). The mixture was stirred for
15 min, and the phases were separated. The organic layer was washed with brine
in water
(1:1, 2 x 50 mL) and the volume of the organic layer was reduced to ¨ 10 mL to
provide
the crude title intermediate.
c. Preparation of 3 -endo- {842-(cyclohexylmethylamino)ethy11-8-aza-
bicyclo[3.2.1]oct-3-
yllbenzamide
To the product of the previous step was added Et0H (30 mL) and concentrated
HC1 (1.5 mL). The solution was purged with nitrogen, 10 % palladium on carbon
(470 mg) was added and the mixture was purged with nitrogen for 5 min and then

hydrogenated at 30 psi overnight. After purging with nitrogen for 2 min, the
solution was
filtered through Celite and solvent was removed to ¨10 mL. Isopropyl acetate
(40 mL)
and 1 M NaOH (20 mL) were added. The layers were separated and the organic
layer was
washed with brine (20 mL), phases were separated and organic solvent removed
to 5-10
mL. Isopropyl acetate (20 mL) was added and the volume reduced to ¨ 8 mL to
which
isopropyl acetate (20 mL) was added. The resulting slurry was stirred at room
temperature for 2 h. The product was isolated by filtration, the reaction
flask and filter
cake were washed with isopropyl acetate (10 mL) to yield the title
intermediate (2.4 g,
98 % pure) as an off-white solid.
d. Preparation of 3 -endo-(8- {2-[cyclohexylmethyl-((S)-2,3-dihydroxy-
propionypaminoJethyl)-8-aza-bicyclo[3.2.1]oct-3-yObenzamide sulfate (hydrate
form)
To a 500 mL flask was added 3 -endo- {8-[2-(cyclohexylmethylamino)ethyl]-8-aza-

bicyclo[3.2.1]oct-3-yl}benzamide (31 g, 83.9 mmol) and DMF (150 mL). The
mixture
was stirred for 10 min and then benzotriazol-1-yloxytris(pyrrolidino)-
phosphonium
hexafluoro-phosphate (56.8 g, 109 mmol) and lithium (45)-2,2-dimethy1-1,3-
dioxolane-4-
carboxylate (15.6 g, 92.3 mmol) were added and the mixture was stirred at room

temperature for 2 h. Ethyl acetate (600 mL) and 0.5 M NaOH (300 mL) were added
and
the phases were separated. The organic layer contained crude (5)-2,2-dimethyl-
[1,3]dioxolane-4-carboxylic acid {243-(3-carbamoyl-pheny1)-8-
azabicyclo[3.2.1]oct-8-
yl]ethyl}cyclohexylmethyl-amide (-84 mmol) which was not isolated.
31

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The organic layer was washed with brine in water (1:1, 2 x 300 mL) and the
phases were separated. To the organic layer was added 2 M H2SO4 (42 mL) and
the
reaction mixture was stirred at room temperature overnight. Acetonitrile (300
mL) was
added and the resulting slurry was stirred for 2 h. The product was isolated
by filtration,
the filter cake was washed with acetonitrile (200 mL), dried in air for 2 h
and then under
vacuum at room temperature for 20 h to provide the title compound (40 g, 97 %
pure by
HPLC) as a white powder.
e. Synthesis of crystalline 3-endo-(8- {2-{cyclohexylmethyl4S)-2,3-dihydroxy-
propionyflaminolethyll-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide sulfate
To a 100 mL flask was added 3-endo-(8- {2-[cyclohexylmethyl-((5)-2,3-
dihydroxy-propionyl)amino]ethy1}-8-aza-bicyclo[3.2.1]oct-3-yObenzamide sulfate

hydrate form (2 g) and Me0H (40 mL). The resulting slurry was heated to 65 C
under
nitrogen for 20 min resulting in complete dissolution. The solution was cooled
to room
temperature with stirring. About 20 mL of solvent was removed under slightly
reduced
pressure and the resulting slurry stirred at room temperature overnight. The
product was
isolated by filtration, and the flask and filter cake were washed with
acetonitrile
(2 x 5 mL). The filter cake was dried in air for 2 h and then under vacuum at
room
temperature overnight to provide the title compound (1.71 g, >99 % pure by
HPLC,
¨85 % yield) as a white powder.
A sample prepared according to the above procedure was characterized by
IHNMR (400 MHz, DMSO d6): 8 (ppm) 9.08 & 8.94 (two sets of brs, 1H), 7.99-8.04

(m, 2H), 7.74-7.76 (m, 111), 7.68-7.70 (m, 1H), 7.41-7.45 (m, 2H), 4.81, 5.00
and 5.30
(three sets of brs, 2H), 4.34 (deformed m, 1H), 4.00 & 4.05 (deformed m, 2H),
3.01-3.25
and 3.47-3.55 and 3.75-3.82 (three sets of m, 10H), 2.50-2.55 (m, 2H), 1.99
(deformed m,
2H), 1.56-1.70(m, 8H), 1.15-1.19 (m, 3H), 0.89-0.99 (m, 2H).
Example 1B : Synthesis of crystalline 3-endo-(8-12-[cyclohexylmethyl4S)-2,3-
dihydroxy-propionyl)aminojethyl}-8-aza-bicyclo[3.2.11oct-3-yl)benzamide
sulfate
a. Preparation of 3 -endo-(8- {2-rcyclohexylmethy1-0)-2,3-dih_ydroxy-
propionyflaminolethy11-8-aza-bicyclo.[3.2.1]oct-3-yl)benzamide sulfate
A mixture of 3 -endo- {842-(cyclohexylmethylamino)ethy1]-8-aza-
bicyclo[3.2.1]oct-3-yl}benzamide (100 g, 270.6 mmol) and DMF (480 mL) was
stirred
for 10 min and then cooled at 0 C. Benzotriazol-1-
yloxytris(pyrrolidino)phosphonium
32

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hexafluorophosphate (183 g, 352 mmol) and lithium (4S)-2,2-dimethy1-1,3-
dioxolane-4-
carboxylate (49.3 g, 324 mmol) were added in one portion at 0 C. The reaction
mixture
was stirred at room temperature for 6 h. Isopropyl acetate (2.0 L) and 1 M
NaOH (1.0 L)
were added, the reaction mixture was stirred for 15 min, and the phases were
separated.
The organic layer was washed with brine in water (1:1, 2 x 1.0 L) and the
phases were
separated. The organic layer was reduced to a quarter of the volume (-500 mL),

acetonitrile (500 mL) was added and the reaction mixture was stirred until
homogenous to
provide a solution of intermediate (S)-2,2-dimethy1-1,3-dioxolane-4-carboxylic
acid 243-
endo-(3-carbamoyl-pheny1)-8-azabicyclo[3.2.1]oct-8-y11-ethyl-cyclohexylmethyl-
amide in
isopropyl acetate and acetonitrile.
An aliquot of the above solution of intermediate (3.03 g, 6.09 mmol) in
isopropyl
acetate/acetonitrile (22.5 mL ) was combined with 2.0 M sulfuric acid in water
(3.68 mL)
and held at 25 C for 20 h and then held at 10 C with stirring for 5 h. The
reaction
solution was filtered, and the filter cake was washed with acetonitrile (25
mL) and dried
to yield the intermediate grade sulfate salt of the title compound (2.91 g,
99.4% purity by
HPLC) as a white solid, predominantly in crystalline hydrate form.
b. Synthesis of crystalline 3-endo-(8- {24cyclohexylmethyl-((S)-2,3-dihydroxy-
propionybaminolethyl}-8-aza-bicyclo[3.2.1]oct-3-ylThenzamide sulfate
A mixture of 3-endo-(8-2-[cyclohexylmethyl-((S)-2,3-dihydroxy-propiony1)-
amino]-ethy1-8-aza-bicyclo[3.2.1]oct-3-y1)-benzamide sulfate intermediate
grade (154.0 g,
277.1 mmol) prepared as in the previous step and methano1/10 % water (616 mL)
was
heated at 65 C over 45min with stirring. The reaction mixture was cooled to
55 C, seed
crystals of the title product (120 mg) were added, the reaction mixture was
stirred at 55 C
for 1 h, and the temperature was reduced to 20 C at the rate of 10 C/h and
then held for
8 h. The reaction mixture was cooled to 5 C, held for 30 min, and filtered.
The filter
cake was washed with methanol (2 x 25 mL) and dried overnight under high
vacuum to
yield the title compound (126.3 g, 99.9 % purity)
Example 2 : Recrystallization of 3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)aminolethyll-8-aza-bicyclo[3.2.1loct-3-yl)benzamide
sulfate (hydrate form)
Compound 1 sulfate (hydrate form) (920 mg) was suspended in acetonitrile (5
mL)
and heated to 65 C. Water (2.4 mL) was then added dropwise until complete
dissolution
was achieved. The resulting solution was cooled to ambient temperature over 20
min.
33

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Nucleation was observed around 35 C. The solids were isolated by vacuum
filtration,
washed with acetonitrile (5 mL) and dried to provide the title compound.
Example 3 : Crystallization of 3-endo-(8-12-[cyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)aminoJethyl}-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide
sulfate
Compound 1 sulfate (hydrate form) (50 mg) was dispersed in a water (10 %) and
methanol (90 %) solvent mixture (0.83 mL) and heated to 60 C with stirring.
The
resulting solution was allowed to cool to ambient temperature over 2 h. The
resulting
solids were isolated by vacuum filtration to provide the title compound (8
mg).
Example 4 : Crystallization of 3-endo-(8-12-[cyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)amino]ethyl}-8-aza-bicyclo[3.2.11oct-3-yl)benzamide
sulfate
Compound 1 sulfate (hydrate form) (42 mg) was dispersed in a water (25 %) and
methanol (75 %) solvent mixture (0.42 mL) and heated to 60 C with stirring.
The
resulting solution was allowed to cool to ambient temperature. The volume was
reduced
by 50 % by rotary evaporation and the solution was left at ambient temperature
overnight.
The resulting solids were isolated by vacuum filtration to provide the title
compound
(8 mg).
Example 5 : Crystallization of 3-endo-(8-{2-Icyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)aminolethyl}-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide
sulfate
Compound 1 (11 mg) was dissolved in a toluene (22 %) and acetonitrile (78 %)
solvent mixture (0.2 mL). Acetonitrile (0.15 mL) was added, followed by 0.04 M
sulfuric
acid in acetonitrile (0.59 mL). A solid precipitate formed on addition of
acid. The
reaction mixture was left at ambient temperature for 12 h. The resulting
solids were
isolated by filtration to provide the title compound.
Example 6 : Crystallization of 3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-
dihydroxy-propionypaminolethyl}-8-aza-bicyclo[3.2.11oct-3-y1)benzamide
sulfate
Compound 1 (38 mg) was dissolved in dichloromethane (0.5 mL). To the solution
was added 0.04 M sulfuric acid in acetonitrile (1.91 mL). A solid precipitate
formed on
addition of acid. The reaction mixture was left at ambient temperature for 12
h. The
resulting solids were isolated by filtration to provide the title compound.
34

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Example 7 : Crystallization of 3-endo-(8-{2-[cyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)aminolethyll-8-aza-bicyclo[3.2.1]oct-3-y1)benzamide
sulfate
Compound 1 (22 mg) was dissolved in a toluene (23 %) and acetonitrile (77 %)
solvent mixture (0.41 mL). To the solution was added 0.04 M sulfuric acid in
acetonitrile
(1.20 mL). A solid precipitate formed on addition of acid. Water (0.16 mL) was
added to
the reaction mixture dissolving the precipitate. Nucleation was observed after
2 h. The
resulting solids were isolated by vacuum filtration to provide the title
compound.
Example 8 : Crystallization of 3-endo-(8-{2-Icyclohexylmethyl-((S)-2,3-
dihydroxy-propionyl)aminolethyll-8-aza-bicyclo[3.2.11oct-3-yl)benzamide
sulfate (hydrate form)
Crystalline compound 1 sulfate (7.1 g) was dissolved in a solvent mixture of
water
(42 mL) and acetonitrile (25 mL). The solution was lyophilized to produce an
amorphous
sulfate salt. The amorphous salt (6.6 g) was dispersed in a acetonitrile (75
%) and water
(25 %) solvent mixture (34.6 mL) and heated to 65 C for 10 min with stirring
and
allowed to cool with stirring until ambient temperature was reached. After 12
h, the
resulting solids were isolated by vacuum filtration to provide the title
compound (5.4 g).
Examples 9-17: Properties of Salt Forms of the Invention
Samples of the crystalline sulfate salt of 3-endo-(8- {2-[cyclohexylmethy1-
((5)-2,3-
dihydroxy-propionyl)amino]ethyl)-8-aza-bicyclo[3.2.1]oct-3-yl)benzamide
(compound 1)
prepared as in Example 1 A and of the crystalline hydrate of the sulfate salt
of compound
1, prepared as in Example 2 were analyzed by x-ray powder diffraction (XRPD),
differential scanning calorimetry (DSC), thermogravimetric analysis (TGA)
infrared
spectroscopy (IR) and ion chromatography.
Example 10: X-Ray Powder Diffraction
X-ray powder diffraction patterns of Figures 1 and 4 were obtained with a
Rigaku
diffractometer using Cu Ka (30.0 kV, 15.0 mA) radiation. The analysis was
performed
with the goniometer running in continuous-scan mode of 3 per min with a step
size of
0.03 over a range of 2 to 40 . Samples were prepared on quartz specimen
holders as a
thin layer of powdered material. The instrument was calibrated with a silicon
standard.

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Example 11: Thermal Analysis
Differential scanning calorimetry (DSC) was performed using a TA Instruments
Model Q-100 module. Data were collected and analyzed using TA Instruments
Thermal
Advantage for Q SeriesTM software. A sample of about 1-10 mg was accurately
weighed
into an aluminum pan with lid. The sample was evaluated using a linear heating
ramp of
C/min from 5 C to, typically, 265 C. The DSC cell was purged with dry
nitrogen
during use. Representative DSC traces for samples of the crystalline sulfate
salt and of
the crystalline hydrate of a sulfate salt of compound 1 are shown in Figures 2
and 5,
respectively.
10 Thermogravimetric analysis (TGA) was performed using a TA Instruments
Model
Q-500 module. Data were collected and analyzed using TA Instruments Thermal
Advantage for Q SeriesTM software. A sample weighing about 1-5 mg was placed
in an
aluminum pan on a platinum cradle and scanned from ambient temperature to 300
C
with a linear heating rate of 10 C/min. The balance and furnace chambers were
purged
with nitrogen during use. Representative TGA traces for samples of a
crystalline sulfate
salt and of a crystalline hydrate of a sulfate salt of compound 1 are also
shown in Figures
2 and 5, respectively.
Example 12: Dynamic Moisture Sorption Assessment
Dynamic moisture sorption (DMS) assessment was performed at 25 C using a
VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, FL 33016). A
sample size of approximately 5-10 mg was used and the humidity was set at the
ambient
value at the start of the analysis. A typical DMS analysis consisted of three
scans:
ambient to 2% relative humidity (RH), 2% RH to 90% RH, 90% RH to 5% RH at a
scan
rate of 5 % RH/step. The mass was measured every two minutes and the RH was
changed
to the next value ( 5 %RH) when the mass of the sample was stable to within
0.02 % for
5 consecutive points. Representative DMS traces for samples of a crystalline
sulfate salt
and of a crystalline hydrate of a sulfate salt of compound 1 are shown in
Figures 3 and 6,
respectively.
Example 13: Infrared Analysis
The infrared (IR) absorption spectrum was determined over the frequency range
4000 to 400 cm-1 using an Avatar 360 FT-IR spectrometer equipped with a
diffuse
reflectance infrared fourier transform spectroscopy (DRIFTS) module. A
representative
36

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IR absorption spectrum for a sample of a crystalline sulfate salt of the
invention had
significant absorption bands at 430 1, 590 1, 639 II, 705 1, 867 1, 1036
1,
1053 1, 1105 1, 1171 1, 1231 1, 1277 1, 1375 1, 1391 1, 1452 1, 1476
1, 1553
1, 1596 1, 1639 1, 1664 1, 2852 . 1, 2907 1, 2928 1, 2967 1, 3168 1,
and
3357 1
Example 14: X-ray Diffraction Crystal Structure Analysis
A chunk crystal of the sulfate salt of compound 1 having dimensions of
0.43 x 0.05 x 0.031 mm was mounted on a glass fiber. X-ray diffraction crystal
structure
TM
data was obtained using a Bniker SMART 6K CCD x¨ray area detector with window
TM
diameter of 13.5 cm, controlled by SMART version 5.630 software (Bruker, 2003)
using
Cu Ka radiation. The sample to detector distance was 5.039 cm. Data was
collected at a
TM
temperature of -153 1 C and was analyzed using SHELXS version 6.14 (Bruker,
2003)
software. The following lattice parameters were derived: unit cell is
orthorhombic with
dimensions a = 6.8239 A, b = 16.2275 A, c = 24.2021 A, a = (I = y = 900; cell
volume
(V) of 2680.0 A3; calculated density is 1.38 g/cm3; space group is
P212121(#19) Powder
x-ray diffraction peaks predicted from the derived atomic positions according
to Mercury
1.4 software were judged by visual inspection to be in excellent agreement
with the
experimental results of Figure 1.
Example 15: Solid State Stability Assessment
Samples of the sulfate salt of the invention were stored in multiple open
glass
vials at 20 C and 60 % relative humidity (RH) and at 40 C and 75 % RH. At
specific
intervals, the contents of a representative vial was removed and analyzed by
DSC, TGA,
PXRD, and by HPLC for chemical purity. After 4 weeks of storage, there was no
detectable change in the DSC or TGA thennograms nor in the XRPD pattern of
samples
stored at either condition. The chemical purity of the stored samples by HPLC
was
unchanged at 99.7 %.
Example 16: Determination of Counterion Content
A sample of the sulfate salt of the invention was analyzed by sulfate ion
TM
chromatography using a Dionex ICS-2000 ion chromatography system equipped with
an
anion self-regenerating suppressor, conductivity detector, IonPac AS11-HC
analytical
TM
anion-exchange column, and IonPac AG11-HC guard column. The sulfate content of
the
37

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sample was determined to be 17.1 % which may be compared with a theoretical
sulfate
content of 17.6 % for one molar equivalent of sulphate ion per mole of parent
compound.
Example 17: Determination of Water Content of Hydrate
A sample of the hydrate of the invention was analyzed by TGA coupled with IR
analysis of the material vaporized during initial weight loss. The TGA trace
shows a
weight loss of 3.2 % below 100 C which may be compared with a theoretical
weight loss
of 3.1 % for a monohydrate of compound 1 sulfate. The JR spectrum of the
vaporized
material was consistent with the reference ER spectrum of water.
Assay 1: Radioligand Binding Assay on Human Mu, Human Delta and
Guinea Pig Kappa Opioid Receptors
a. Membrane Preparation
CHO-K 1 (Chinese Hamster Ovary) cells stably transfected with human mu opioid
or with guinea pig kappa receptor cDNA were grown in medium consisting of
Ham's-F12
media supplemented with 10% FBS, 100 units/ml penicillin - 100 pg/mL
streptomycin
TM
and 800 p.g/mL Geneticin in a 5% CO2, humidified incubator @ 37 C. Receptor
expression levels (B,õ.õ ¨2.0 and ¨0.414 pmol/mg protein, respectively) were
determined
using [31-1]-Diprenorphine (specific activity ¨ 50-55 Ci/mmol) in a membrane
radioligand
binding assay.
Cells were grown to 80-95% confluency (< 25 subculture passages). For cell
line
passaging, the cell monolayer was incubated for 5 minutes at room temperature
and
harvested by mechanical agitation in 10 mL of PBS supplemented with 5 mM EDTA.

Following resuspension, cells were transferred to 40 mL fresh growth media for

centrifugation for 5 minutes at 1000 rpm and resuspended in fresh growth
medium at the
appropriate split ratio.
For membrane preparation, cells were harvested by gentle mechanical agitation
with 5 mM EDTA in PBS followed by centrifugation (2500 g for 5 minutes). The
pellets
were resuspended in Assay Buffer (50 mM 4-(2-hydroxyethyppiperazine-1-
ethanesulfonic
acid N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)), pH 7.4,
and
homogenized with a polytron disrupter on ice. The resultant homogenates were
centrifuged (1200 g for 5 minutes), the pellets discarded and the supernatant
centrifuged
(40,000 g for 20 minutes). The pellets were washed once by resuspension in
Assay
Buffer, followed by an additional centrifugation (40,000 g for 20 minutes).
The final
38

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pellets were resuspended in Assay Buffer (equivalent 1 T-225 flask/1 mL assay
buffer).
TM
Protein concentration was determined using a Bio-Rad Bradford Protein Assay
kit and
membranes were stored in frozen aliquots at -80 C, until required.
Human delta opioid receptor (hDOP) membranes were purchased from Perkin
TM
Elmer. The reported IQ and Bmax for these membranes determined by saturation
analyses
in a [311]-Natrindole radioligand binding assays were 0.14 nIVI (pIQ = 9.85)
and
2.2 pmol/mg protein, respectively. Protein concentration was determined using
a Bio-Rad TM
Bradford Protein Assay kit. Membranes were stored in frozen aliquots at -80
C, until
required.
b. Radioligand Binding Assays
TM
Radioligand binding assays were performed in an Axygen 1.1 mL deep well
96-well polypropylene assay plate in a total assay volume of 200 1.,
containing the
appropriate amount of membrane protein (-3, ¨2 and ¨20 ptg for mu, delta and
kappa,
respectively) in Assay Buffer, supplemented with 0.025% bovine serum albumin
(BSA).
Saturation binding studies for determination of Kd values of the radioligand
were
performed using [311]-Diprenorphine at 8-12 different concentrations ranging
from 0.001
nM ¨ 5 nM. Displacement assays for determination of pKi values of compounds
were
performed with [311]-Diprenorphine at 0.5, 1.2, and 0.7 nM for mu, delta, and
kappa,
respectively, and eleven concentrations of compound ranging from 10 pM ¨ 100
M.
TM
Binding data were analyzed by nonlinear regression analysis with the GraphPad
Prism Software package (GraphPad Software, Inc., San Diego, CA) using the
3-parameter model for one-site competition. The curve minimum was fixed to the
value
for nonspecific binding, as determined in the presence of 101.1M naloxone. Ki
values for
test compounds were calculated, in Prism, from the best fit IC50 values, and
the IQ value
of the radioligand, using the Cheng-Prusoff equation gq-- +([1.,pcdp where
[L,) =
the concentration of [31-1]-Diprenorphine. Results are expressed as the
negative decadic
logarithm of the Ki values, pKi.
Test compounds having a higher pKi value in these assays have a higher binding

affinity for the mu, delta, or kappa opioid receptor. The sulfate salt of
compound 1
exhibited a pKi value of 9.9 at the human mu opioid receptor.
39

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Assay 2: Agonist mediated activation of the mu-opioid receptor in
membranes prepared from CHO-Kl cells expressing the human mu-opioid
receptor
In this assay, the potency and intrinsic activity values of test compounds
were
determined by measuring the amount of bound GTP-Eu present following receptor
activation in membranes prepared from CHO-Kl cells expressing the human mu
opioid
receptor.
a. Mu Opioid Receptor Membrane Preparation:
Human mu opioid receptor (hM0P) membranes were either prepared as described
above or were purchased from Perkin Elmer. The reported plc! and BT.. for the
purchased membranes determined by saturation analyses in a [311]-Diprenorphine

radioligand binding assays was 10.06 and 2.4 pmol/mg protein, respectively.
Protein
concentration was determined using a Bio-Rad Bradford Protein Assay kit.
Membranes
were stored in frozen aliquots at -80 C, until required. Lyophilized GTP-Eu
and GDP
were diluted to10 tiM and 2 mM, respectively, in double distilled H20 then
mixed and
permitted to sit at room temperature for 30 minutes prior to transfer to
individual aliquots
samples for storage at ¨20 C.
b. Human mu GTP-Eu nucleotide exchange assay
TM
GTP-Eu nucleotide exchange assays were performed using the DELPHIA GTP-
TM TM
binding kit (Perkin/Elmer) in AcroWell 96 well filter plates according to the
manufacturer's specifications. Membranes were prepared as described above, and
prior to
the start of the assay, aliquots were diluted to a concentration of 200 glmL
in Assay
Buffer (50mM HEPES, pH 7.4 at 25 C), then homogenized for 10 seconds using a
TM
Polytron homogenizer. Test compounds were received as 10 mM stock solutions in
DMSO, diluted to 400 M into Assay Buffer containing 0.1% BSA, and serial
(1:5)
dilutions then made to generate ten concentrations of compound ranging from 40
pM -
80 M - GDP and GTP-Eu were diluted to 4 M and 40 nM, respectively, in Assay
Buffer. The assay was performed in a total volume of 100 I, containing 5 pig
of
membrane protein, test compound ranging from 10 pM ¨ 20 M), 1 LIM GDP, and 10
nM
GTP-Eu diluted in 10 mM MgC12, 50 mM NaC1, and 0.0125% BSA, (final assay
concentrations). A DAMGO (Tyr-D-Ala-Gly-(methyl)Phe-Gly-ol) concentration-
response curve (ranging from 12.8 pM ¨ 1 M) was included on every plate.

CA 02678073 2014-05-13
WO 2008/106159 PCT/US2008/002583
Assay plates were prepared immediately prior to assay following the addition
of
251.1L of Assay Buffer, 25 tL of test compound, and 251.1.1, GDP and GTP-Eu.
The assay
was initiated by the addition of 25 juL membrane protein and allowed to
incubate for
TM
30 minutes. The assay plates were then filtered with a Waters vacuum manifold
connected to the house vacuum regulated to 10-12 in. Hg and washed with room
temperature GTP Wash Solution (2 x 300 mL). The bottoms of the plates were
blotted to
remove excess liquid. The plates were then immediately read to determine the
amount of
TM
bound GTP-Eu by measuring Time Resolved Fluorescence (TRF) on a Packard Fusion

Plate ReaderVehicle: DMSO not to exceed I% final assay concentration.
The amount of bound GTP-Eu is proportional to the degree of activation of the
mu
opioid receptors by the test compound. The intrinsic activity (IA), expressed
as a
percentage, was determined as the ratio of the amount of bound GTP-Eu observed
for
activation by the test compound to the amount observed for activation by DAMGO
which
is presumed to be a full agonist (IA=100). The sulfate salt of compound 1
demonstrated
an intrinsic activity of -5 in this assay. Thus, the present sulfate salt has
been shown to be
an antagonist.
Assay 3: Rat Model of In Vivo Efficacy
In this assay the efficacy of test compounds was evaluated in a model of
gastrointestinal transit, which evaluates peripheral activity. This study was
approved by
the Institutional Animal Care and Use Committee at Theravance, Inc. and
conformed to
the Guide for the Care and Use of Laboratory Animals published by the National
Academy of Sciences (01996).
a. Rat Gastric Emptying Assay
Test compounds were evaluated in the rat gastric emptying assay to determine
their ability to reverse loperamide-induced delayed gastric emptying. Rats
were fasted up
overnight prior to administration of test compounds or vehicle by intravenous,

subcutaneous, intramuscular or oral routes of administration at doses ranging
from 0.001
to about 30 milligrams/kilogram (mg/kg). The administration of test compound
was
followed by subcutaneous administration of loperamide at a dose of 1 mg/kg or
vehicle.
Five minutes post loperamide or vehicle administration, a non-nutritive, non-
absorbable
charcoal meal was administered via oral gavage and animals were allowed free
access to
water for the sixty minute duration of the experiment. Animals were then
euthanized via
41

CA 02678073 2015-01-22
,
,
carbon dioxide asphyxiation followed by thoracotomy and the stomach was
carefully
excised. The stomach was ligated at the lower esophageal sphincter and the
pyloric
sphincter to prevent additional emptying during tissue removal. Gastric weight
was then
determined after removal of the ligatures.
b. Data Analysis and Results
Data was analyzed using the GraphPad Prism Software package (GraphPad
Software, Inc., San Diego, CA). Percent reversal curves were constructed by
non-linear
regression analysis using the sigmoidal dose response (variable slope) model
and best-fit
ID50 values were calculated. Curve minima and maxima were fixed to loperamide
control
values (indicating 0% reversal) and vehicle controls (indicating 100%
reversal),
respectively. Results are expressed as Ipso, the dose required for 50%
reversal of the
effects of loperamide, in milligrams per kilogram. The sulfate salt of
compound 1,
administered orally, exhibited an lD50 value of 0.26 mg/kg in the gastric
emptying model.
The scope of the claims should not be limited by the preferred embodiments
set forth in the examples, but should be given the broadest interpretation
consistent with the
description as a whole.
42